Methods and compositions for improving efficiency of nucleic acids amplification reactions

ABSTRACT

The present invention provides methods and compositions for improving the efficiency of nucleic acid amplification reactions. The invention encompasses hybrid polymerases that show increased processivity over wild type polymerases as well as decreased exonucleases activity. The invention also encompasses methods, compositions and kits for conducting nucleic acid synthesis and amplification reactions in which non-specific amplification of primers is reduced.

CROSS-REFERENCES TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 14/482,756, filed Sep. 10, 2014, which is a continuation ofU.S. patent application Ser. No. 12/683,950, filed Jan. 7, 2010, nowU.S. Pat. No. 8,859,205, which claims benefit of priority to U.S.Provisional Patent Application No. 61/143,350, filed Jan. 8, 2009, eachof which is incorporated by reference in its entirety for all purposes.

REFERENCE TO SUBMISSION OF A SEQUENCE LISTING AS A TEXT FILE

The Sequence Listing written in file SEQTXT_094260-1087961.txt, createdon May 7, 2018, 34,217 bytes, machine format IBM-PC, MS-Windowsoperating system, is hereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to nucleic acid amplification reactions.The present invention encompasses hybrid polymerases with improvedprocessivity as well as methods and compositions for reducingnon-specific amplification during reactions such as PCR.

BACKGROUND OF THE INVENTION

Nucleic acid amplification reactions, such as polymerase chain reaction(PCR), are generally template-dependent reactions in which a desirednucleic acid sequence is amplified by treating separate complementarystrands of a target nucleic acid with an excess of two oligonucleotideprimers. The primers are extended to form complementary primer extensionproducts which act as templates for synthesizing the desired nucleicacid sequence. In such processes, the nucleic acid sequence between theprimers on the respective DNA strands is selectively amplified.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention provides methods and compositions forimproving the efficiency and the specificity of nucleic acid synthesisand amplification reactions.

In one aspect, the present invention provides a method of amplifying atarget nucleic acid. This method includes incubating the target nucleicacid with a reaction mixture that includes at least one primer, a hybridpolymerase, and an osmolyte. In a further aspect, the incubating step isconducted under conditions that permit amplification of the targetnucleic acid by the hybrid polymerase. In a still further aspect, thehybrid polymerase comprises a polymerase domain and a DNA bindingdomain.

In a further aspect, the invention provides a kit for quantitative PCR.In an exemplary aspect, the kit includes: a hybrid polymerase withreduced exonuclease activity, dNTPs, a buffer that includes sarcosine,and directions for using the hybrid polymerase in a nucleic acidamplification reaction.

In some embodiments, the invention provides for a method of amplifying atarget nucleic acid in a sample. In some embodiments, the methodcomprises

incubating said target nucleic acid in a reaction mixture comprising:

-   -   at least one primer,    -   a hybrid polymerase comprising a polymerase domain and a        heterologous DNA binding domain, and    -   an additive selected from the group consisting of sarcosine and        heparin, in a sufficient amount to improve efficiency of the        amplification reaction by at least 10% compared to a reaction        mixture lacking the additive,        wherein said incubating is under conditions that permit        amplification of said target nucleic acid by said hybrid        polymerase.

In some embodiments, the additive is sarcosine, which is at aconcentration of less than 600 mM.

In some embodiments, the hybrid polymerase comprises a mutation in anexonuclease domain such that the polymerase substantially lacks a 3′-5′exonuclease activity. In some embodiments, the hybrid polymerasecomprises a Family B-like polymerase that substantially lacks (e.g., hasa mutation resulting in a substantial lack of) a 3′-5′ exonucleaseactivity. In some embodiments, the hybrid polymerase comprises apolypeptide sequence that is substantially identical to SEQ ID NO:2,i.e., is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% or greater amino acid sequence identity across thepolymerase domain (i.e., all but SEQ ID NO:3) or the entire sequence ofSEQ ID NO:2. In some embodiments, the hybrid polymerase comprises SEQ IDNO:2.

In some embodiments, the sample comprises an amplification inhibitor andwherein an aliquot of the sample is added to the reaction mixture in anamount such that the inhibitor is at a concentration capable ofinhibiting wildtype Taq polymerase and wherein the hybrid polymeraseamplifies the target nucleic acid. In some embodiments, the sample isselected from blood or intact cells or food samples.

In some embodiments, the hybrid polymerase is complexed with one or moreantibodies prior to a heating step during the incubating step. In someembodiments, the antibody comprises a heavy chain variable region and alight chain variable region, wherein the variable regions comprisecomplementary determining regions (CDRs), wherein:

-   -   (a) the heavy chain variable region CDRs comprise SEQ ID NO:22,        SEQ ID NO:23, and SEQ ID NO:24, and the light chain variable        region CDRs comprise SEQ ID NO:25, SEQ ID NO:26, and SEQ ID        NO:27; or    -   (b) the heavy chain variable region CDRs comprise SEQ ID NO:28,        SEQ ID NO:29, and SEQ ID NO:30, and the light chain variable        region CDRs comprise SEQ ID NO:31, SEQ ID NO:32, and SEQ ID        NO:33; or    -   (c) the heavy chain variable region CDRs comprise SEQ ID NO:34,        SEQ ID NO:35, and SEQ ID NO:36, and the light chain variable        region CDRs comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID        NO:39.

In some embodiments, signal from the amplification is detected inreal-time.

In some embodiments, the heterologous DNA binding domain issubstantially identical to (i.e., at least 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater) to SEQ IDNO:3.

In some embodiments, the amplification reaction is performed withextension times of less than 1, 2, 3, or 4 seconds.

In some embodiments, the polymerase and exonuclease domains are from aFamily B DNA polymerase.

In some embodiments, the reaction mixture further comprises adouble-stranded DNA binding dye.

The present invention also provides for reaction mixtures. In someembodiments, the reaction mixture comprises:

a hybrid polymerase, wherein said polymerase comprises a polymerasedomain and a heterologous DNA binding domain; and

an additive selected from the group consisting of sarcosine and heparin,in a sufficient amount to improve efficiency of the amplificationreaction by at least 10% compared to a reaction mixture lacking theadditive.

In some embodiments, the reaction mixture further comprises at least oneoligonucleotide primer.

In some embodiments, the additive is sarcosine, which is at aconcentration of less than 600 mM.

In some embodiments, the hybrid polymerase comprises a mutation in anexonuclease domain such that the polymerase substantially lacks a 3′-5′exonuclease activity. In some embodiments, the hybrid polymerasecomprises a Family B-like polymerase that substantially lacks (e.g., hasa mutation resulting in a substantial lack of) a 3′-5′ exonucleaseactivity. In some embodiments, the hybrid polymerase comprises apolypeptide sequence that is substantially identical to SEQ ID NO:2,i.e., is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% or greater amino acid sequence identity across thepolymerase domain (i.e., all but SEQ ID NO:3) or the entire sequence ofSEQ ID NO:2. In some embodiments, the polymerase comprises SEQ ID NO:2.

In some embodiments, the mixture comprises an aliquot of a sample,wherein said sample comprises an amplification inhibitor, wherein theinhibitor is at a concentration capable of inhibiting wildtype Taqpolymerase. In some embodiments, the sample is selected from blood orintact cells or food samples.

In some embodiments, the hybrid polymerase is complexed with anantibody.

In some embodiments, the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the variable regionscomprise complementary determining regions (CDRs), wherein:

-   -   (a) the heavy chain variable region CDRs comprise SEQ ID NO:22,        SEQ ID NO:23, and SEQ ID NO:24, and the light chain variable        region CDRs comprise SEQ ID NO:25, SEQ ID NO:26, and SEQ ID        NO:27; or    -   (b) the heavy chain variable region CDRs comprise SEQ ID NO:28,        SEQ ID NO:29, and SEQ ID NO:30, and the light chain variable        region CDRs comprise SEQ ID NO:31, SEQ ID NO:32, and SEQ ID        NO:33; or    -   (c) the heavy chain variable region CDRs comprise SEQ ID NO:34,        SEQ ID NO:35, and SEQ ID NO:36, and the light chain variable        region CDRs comprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID        NO:39.

In some embodiments, the heterologous DNA binding domain issubstantially identical to (i.e., at least 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater) to SEQ IDNO:3.

In some embodiments, the polymerase and exonuclease domains are from aFamily B DNA polymerase.

In some embodiments, the reaction mixture comprises one or moredifferent deoxyribonucleotide triphosphates.

In some embodiments, the reaction mixture further comprises adouble-stranded DNA binding dye.

The present invention also provides kits. In some embodiments, the kitcomprises:

a hybrid polymerase comprising a polymerase domain and a heterologousDNA binding domain; and

an additive selected from the group consisting of sarcosine and heparin,in a sufficient amount to improve efficiency of an amplificationreaction by at least 10% compared to a reaction mixture lacking theadditive.

In some embodiments, the hybrid polymerase comprises a mutation in anexonuclease domain such that the polymerase substantially lacks a 3′-5′exonuclease activity. In some embodiments, the hybrid polymerasecomprises a Family B-like polymerase that substantially lacks (e.g., hasa mutation resulting in a substantial lack of) a 3′-5′ exonucleaseactivity. In some embodiments, the hybrid polymerase comprises apolypeptide sequence that is substantially identical to SEQ ID NO:2,i.e., is at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% or greater amino acid sequence identity across thepolymerase domain (i.e., all but SEQ ID NO:3) or the entire sequence ofSEQ ID NO:2. In some embodiments, the hybrid polymerase comprise SEQ IDNO:2.

In some embodiments, the kit further comprises an antibody that isspecific for the hybrid polymerase.

In some embodiments, the antibody comprises a heavy chain variableregion and a light chain variable region, wherein the variable regionscomprise complementary determining regions (CDRs), wherein:

(a) the heavy chain variable region CDRs comprise SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:24, and the light chain variable region CDRscomprise SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or

(b) the heavy chain variable region CDRs comprise SEQ ID NO:28, SEQ IDNO:29, and SEQ ID NO:30, and the light chain variable region CDRscomprise SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; or

(c) the heavy chain variable region CDRs comprise SEQ ID NO:34, SEQ IDNO:35, and SEQ ID NO:36, and the light chain variable region CDRscomprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

In some embodiments, the heterologous DNA binding domain issubstantially identical to (i.e., at least 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater) to SEQ IDNO:3.

In some embodiments, the polymerase and exonuclease domains are from aFamily B DNA polymerase.

In some embodiments, the kit further comprises one or more of:

one or more different deoxyribonucleotide triphosphates;

a double-stranded DNA binding dye; and

an oligonucleotide primer.

The present invention also provides for an isolated antibody havingbinding specificity for a protein consisting of SEQ ID NO:2, wherein theantibody comprises a heavy chain and a light chain variable region,wherein the variable regions comprise complementary determining regions(CDRs), wherein:

(a) the heavy chain variable region CDRs comprise SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:24, and the light chain variable region CDRscomprise SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27; or

(b) the heavy chain variable region CDRs comprise SEQ ID NO:28, SEQ IDNO:29, and SEQ ID NO:30, and the light chain variable region CDRscomprise SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33; or

(c) the heavy chain variable region CDRs comprise SEQ ID NO:34, SEQ IDNO:35, and SEQ ID NO:36, and the light chain variable region CDRscomprise SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39.

In some embodiments, the heavy chain and light chain variable regionscomprise:

(a) SEQ ID NO:14 and SEQ ID NO:18, respectively;

(b) SEQ ID NO:15 and SEQ ID NO:19, respectively;

(c) SEQ ID NO:16 and SEQ ID NO:20, respectively; or

(d) SEQ ID NO:17 and SEQ ID NO:21, respectively;

The present invention also provides for an isolated polynucleotideencoding a polypeptide comprising a heavy chain or light chain variableregion as discussed above.

The present invention also provides for an antibody complexed with apolymerase, wherein the antibody comprises a heavy chain and/or lightchain variable region as discussed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of sarcosine on efficiency of a qPCR reaction

FIG. 2 shows amplification curves for reactions containing differentconcentrations of heparin to those with no heparin added.

FIG. 3 shows amplification curves for different hybrid polymerases withchange in annealing/extension temperature. Amplification curves for 25pg, 250 pg, and 2500 pg input DNA are displayed.

FIG. 4A shows the amino acid sequence of an exemplary hybrid polymerasesthat includes a DNA binding domain (SEQ ID NO:2). FIG. 4B shows theamino acid sequence of an exemplary DNA binding domain (SEQ ID NO:3).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing devices, formulations and methodologies whichare described in the publication and which might be used in connectionwith the presently described invention.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a polymerase”refers to one agent or mixtures of such agents, and reference to “themethod” includes reference to equivalent steps and methods known tothose skilled in the art, and so forth.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention. It will be apparent to one of skill inthe art that these additional features are also encompassed by thepresent invention.

Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Generally,the nomenclature used herein and the laboratory procedures in cellculture, molecular genetics, organic chemistry, and nucleic acidchemistry and hybridization described below are those well known andcommonly employed in the art. Standard techniques are used for nucleicacid and peptide synthesis. The techniques and procedures are generallyperformed according to conventional methods in the art and variousgeneral references (see generally, Sambrook et al. MOLECULAR CLONING: ALABORATORY MANUAL, 2d ed. (1989) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., which is incorporated herein by reference),which are provided throughout this document. The nomenclature usedherein and the laboratory procedures in analytical chemistry, andorganic synthetic described below are those well known and commonlyemployed in the art. Standard techniques, or modifications thereof, areused for chemical syntheses and chemical analyses.

The term “hybrid polymerase” is used herein to describe a polymerasethat comprises amino acid residues from multiple parent sequences.Hybrid polymerases may comprise both polymerase domains as well as DNAbinding domains.

The term “hybrid position” refers to a position that differs betweenparent sequences, or subsequences.

A “wild type polymerase” refers to a naturally occurring polymerase. A“wild type polymerase amino acid sequence” refers to the naturallyoccurring amino acid sequence.

A “native” polymerase sequence refers to a parent polymerase sequence,typically a “wildtype” sequence.

A “parent polymerase sequence” indicates a starting or reference aminoacid or nucleic acid sequence prior to a manipulation of the invention.The term is used interchangeably with “starting sequence”. Parentsequences may be wild-type proteins, proteins containing mutations, orother engineered proteins. Parent sequences can also be full-lengthproteins, protein subunits, protein domains, amino acid motifs, proteinactive sites, or any polymerase sequence or subset of polymerasesequences, whether continuous or interrupted by other polypeptidesequences.

The term “DNA binding domain” refers to a protein domain that binds DNAin a sequence non-specific manner. In some embodiments, the DNA bindingdomain is a protein domain which binds with significant affinity to DNA,for which there is no known nucleic acid which binds to the proteindomain with more than 100-fold more affinity than another nucleic acidwith the same nucleotide composition but a different nucleotidesequence.

The term “Sso7d” or “Sso7d DNA binding domain” or “Sso7d-like DNAbinding domain” or “Sso7d binding protein” refers to nucleic acid andpolypeptide polymorphic variants, alleles, mutants, and interspecieshomologs that: (1) have an amino acid sequence that has greater thanabout 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or greater amino acidsequence identity, preferably over a region of at least about 15, 25,35, 50, or 63 amino acids, to an Sso7d sequence of SEQ ID NO: 3; 2) bindto antibodies, e.g., polyclonal antibodies, raised against an immunogencomprising an amino acid sequence of SEQ ID NO: 3 and conservativelymodified variants thereof; (3) specifically hybridize under stringenthybridization conditions to a Sso7d nucleic acid sequence of SEQ ID NO:3 and conservatively modified variants thereof; or (4) have a nucleicacid sequence that has greater than about 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99%, or higher nucleotide sequence identity, preferablyover a region of at least about 50, 100, 150, or more nucleotides, toSEQ ID NO:4. The term includes both full-length Sso7d polypeptides andfragments of the polypeptides that have sequence non-specificdouble-stranded binding activity. Sso7d-like proteins include Sac7d andSac7e.

“Domain” refers to a unit of a protein or protein complex, comprising apolypeptide subsequence, a complete polypeptide sequence, or a pluralityof polypeptide sequences where that unit has a defined function. Thefunction is understood to be broadly defined and can be ligand binding,catalytic activity or can have a stabilizing effect on the structure ofthe protein.

“Efficiency” in the context of a reaction of this invention refers tothe ability of the components of the reaction to perform their functionsunder specific reaction conditions. For example, efficiency may refer tothe ability of a polymerase enzyme to perform its catalytic functionunder specific reaction conditions. Methods for calculating efficiencyof amplification reactions are known in the art and described in furtherdetail herein.

“Enhances” in the context of an enzyme refers to improving the activityof the enzyme, i.e., increasing the amount of product per unit enzymeper unit time.

“Heterologous”, when used with reference to portions of a protein,indicates that the protein comprises two or more domains that are notfound in the same relationship to each other in nature. Such a protein,e.g., a fusion protein, contains two or more domains from unrelatedproteins arranged to make a new functional protein.

“Join” refers to any method known in the art for functionally connectingprotein domains, including without limitation recombinant fusion with orwithout intervening domains, intein-mediated fusion, non-covalentassociation, and covalent bonding, including disulfide bonding; hydrogenbonding; electrostatic bonding; and conformational bonding, e.g.,antibody-antigen, and biotin-avidin associations.

“Polymerase” refers to an enzyme that performs template-directedsynthesis of polynucleotides. The term encompasses both the full lengthpolypeptide and a domain that has polymerase activity.

“Processivity” refers to the ability of a polymerase to remain bound tothe template or substrate and perform polynucleotide synthesis.Processivity is measured by the number of catalytic events (e.g.,nucleotides incorporated) that take place per binding event.

“Thermally stable polymerase” as used herein refers to any enzyme thatcatalyzes polynucleotide synthesis by addition of nucleotide units to anucleotide chain using DNA or RNA as a template and has an optimalactivity at a temperature above 45° C.

“Thermus polymerase” refers to a family A DNA polymerase isolated fromany Thermus species, including without limitation Thermus aquaticus,Thermus brockianus, and Thermus thermophilus; any recombinantpolymerases deriving from Thermus species, and any functionalderivatives thereof, whether derived by genetic modification or chemicalmodification or other methods known in the art.

The term “amplification reaction” refers to any in vitro means formultiplying the copies of a target sequence of nucleic acid. Suchmethods include but are not limited to polymerase chain reaction (PCR),DNA ligase chain reaction (see U.S. Pat. Nos. 4,683,195 and 4,683,202;PCR Protocols: A Guide to Methods and Applications (Innis et al., eds,1990)), (LCR), QBeta RNA replicase, and RNA transcription-based (such asTAS and 3 SR) amplification reactions as well as others known to thoseof skill in the art.

“Amplifying” refers to a step of submitting a solution to conditionssufficient to allow for amplification of a polynucleotide if all of thecomponents of the reaction are intact. Components of an amplificationreaction include, e.g., primers, a polynucleotide template, polymerase,nucleotides, and the like. The term “amplifying” typically refers to an“exponential” increase in target nucleic acid. However, “amplifying” asused herein can also refer to linear increases in the numbers of aselect target sequence of nucleic acid, such as is obtained with cyclesequencing.

The term “amplification reaction mixture” refers to an aqueous solutioncomprising the various reagents used to amplify a target nucleic acid.These include enzymes, aqueous buffers, salts, amplification primers,target nucleic acid, and nucleoside triphosphates. As discussed furtherherein, amplification reaction mixtures may also further includestabilizers and other additives to optimize efficiency and specificity.Depending upon the context, the mixture can be either a complete orincomplete amplification reaction mixture

“Polymerase chain reaction” or “PCR” refers to a method whereby aspecific segment or subsequence of a target double-stranded DNA, isamplified in a geometric progression. PCR is well known to those ofskill in the art; see, e.g., U.S. Pat. Nos. 4,683,195 and 4,683,202; andPCR Protocols: A Guide to Methods and Applications, Innis et al., eds,1990. Exemplary PCR reaction conditions typically comprise either two orthree step cycles. Two step cycles have a denaturation step followed bya hybridization/elongation step. Three step cycles comprise adenaturation step followed by a hybridization step followed by aseparate elongation step.

“Long PCR” refers to the amplification of a DNA fragment of 5 kb or morein length. Long PCR is typically performed using specially-adaptedpolymerases or polymerase mixtures (see, e.g., U.S. Pat. Nos. 5,436,149and 5,512,462) that are distinct from the polymerases conventionallyused to amplify shorter products.

A “primer” refers to a polynucleotide sequence that hybridizes to asequence on a target nucleic acid and serves as a point of initiation ofnucleic acid synthesis. Primers can be of a variety of lengths and areoften less than 50 nucleotides in length, for example 12-30 nucleotides,in length. The length and sequences of primers for use in PCR can bedesigned based on principles known to those of skill in the art, see,e.g., Innis et al., supra.

A “temperature profile” refers to the temperature and lengths of time ofthe denaturation, annealing and/or extension steps of a PCR or cyclesequencing reaction. A temperature profile for a PCR or cycle sequencingreaction typically consists of 10 to 60 repetitions of similar oridentical shorter temperature profiles; each of these shorter profilesmay typically define a two step or three-step cycle. Selection of atemperature profile is based on various considerations known to those ofskill in the art, see, e.g., Innis et al., supra. In a long PCR reactionas described herein, the extension time required to obtain anamplification product of 5 kb or greater in length is reduced comparedto conventional polymerase mixtures.

PCR “sensitivity” refers to the ability to amplify a target nucleic acidthat is present in low copy number. “Low copy number” refers to 10⁵,often 10⁴, 10³, 10², 10¹ or fewer, copies of the target sequence in thenucleic acid sample to be amplified.

The term “polymerase primer/template binding specificity” as used hereinrefers to the ability of a polymerase to discriminate between correctlymatched primer/templates and mismatched primer templates. An “increasein polymerase primer/template binding specificity” in this contextrefers to an increased ability of a polymerase of the invention todiscriminate between matched primer/template and mismatched primercomplexes in comparison to a wild type polymerase.

A “template” refers to a polynucleotide sequence that comprises thepolynucleotide to be amplified, flanked by primer hybridization sites.Thus, a “target template” comprises the target polynucleotide sequenceflanked by hybridization sites for a 5′ primer and a 3′ primer.

As used herein, “nucleic acid” means DNA, RNA, single-stranded,double-stranded, or more highly aggregated hybridization motifs, and anychemical modifications thereof. Modifications include, but are notlimited to, those providing chemical groups that incorporate additionalcharge, polarizability, hydrogen bonding, electrostatic interaction,points of attachment and functionality to the nucleic acid ligand basesor to the nucleic acid ligand as a whole. Such modifications include,but are not limited to, peptide nucleic acids (PNAs), phosphodiestergroup modifications (e.g., phosphorothioates, methylphosphonates),2′-position sugar modifications, 5-position pyrimidine modifications,8-position purine modifications, modifications at exocyclic amines,substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil;backbone modifications, methylations, unusual base-pairing combinationssuch as the isobases, isocytidine and isoguanidine and the like. Nucleicacids can also include non-natural bases, such as, for example,nitroindole. Modifications can also include 3′ and 5′ modifications suchas capping with a fluorophore (e.g., quantum dot) or another moiety.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an artificial chemical mimetic of a corresponding naturallyoccurring amino acid, as well as to naturally occurring amino acidpolymers and non-naturally occurring amino acid polymers.

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, .gamma.-carboxyglutamate, and O-phosphoserine. Aminoacid analogs refers to compounds that have the same basic chemicalstructure as a naturally occurring amino acid, i.e., a carbon atom thatis bound to a hydrogen atom, a carboxyl group, an amino group, and an Rgroup, e.g., homoserine, norleucine, methionine sulfoxide, methioninemethyl sulfonium. Such analogs have modified R groups (e.g., norleucine)or modified peptide backbones, but retain the same basic chemicalstructure as a naturally occurring amino acid. Amino acid mimeticsrefers to chemical compounds that have a structure that is differentfrom the general chemical structure of an amino acid, but that functionsin a manner similar to a naturally occurring amino acid.

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

“Conservatively modified variants” applies to both amino acid andnucleic acid sequences. With respect to particular nucleic acidsequences, conservatively modified variants refers to those nucleicacids which encode identical or essentially identical amino acidsequences, or where the nucleic acid does not encode an amino acidsequence, to essentially identical sequences. Because of the degeneracyof the genetic code, a large number of functionally identical nucleicacids encode any given protein. For instance, the codons GCA, GCC, GCGand GCU all encode the amino acid alanine. Thus, at every position wherean alanine is specified by a codon, the codon can be altered to any ofthe corresponding codons described without altering the encodedpolypeptide. Such nucleic acid variations are “silent variations,” whichare one species of conservatively modified variations. Every nucleicacid sequence herein which encodes a polypeptide also describes everypossible silent variation of the nucleic acid. One of skill willrecognize that each codon in a nucleic acid (except AUG, which isordinarily the only codon for methionine, and TGG, which is ordinarilythe only codon for tryptophan) can be modified to yield a functionallyidentical molecule. Accordingly, each silent variation of a nucleic acidwhich encodes a polypeptide is implicit in each described sequence.

As to amino acid sequences, one of skill will recognize that individualsubstitutions, deletions or additions to a nucleic acid, peptide,polypeptide, or protein sequence which alters, adds or deletes a singleamino acid or a small percentage of amino acids in the encoded sequenceis a “conservatively modified variant” where the alteration results inthe substitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The term “encoding” refers to a polynucleotide sequence encoding one ormore amino acids. The term does not require a start or stop codon. Anamino acid sequence can be encoded in any one of six different readingframes provided by a polynucleotide sequence.

The term “promoter” refers to regions or sequence located upstreamand/or downstream from the start of transcription and which are involvedin recognition and binding of RNA polymerase and other proteins toinitiate transcription.

A “vector” refers to a polynucleotide, which when independent of thehost chromosome, is capable replication in a host organism. Preferredvectors include plasmids and typically have an origin of replication.Vectors can comprise, e.g., transcription and translation terminators,transcription and translation initiation sequences, and promoters usefulfor regulation of the expression of the particular nucleic acid.

“Recombinant” refers to a human manipulated polynucleotide or a copy orcomplement of a human manipulated polynucleotide. For instance, arecombinant expression cassette comprising a promoter operably linked toa second polynucleotide may include a promoter that is heterologous tothe second polynucleotide as the result of human manipulation (e.g., bymethods described in Sambrook et al., Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1989)or Current Protocols in Molecular Biology Volumes 1-3, John Wiley &Sons, Inc. (1994-1998)) of an isolated nucleic acid comprising theexpression cassette. In another example, a recombinant expressioncassette may comprise polynucleotides combined in such a way that thepolynucleotides are extremely unlikely to be found in nature. Forinstance, human manipulated restriction sites or plasmid vectorsequences may flank or separate the promoter from the secondpolynucleotide. One of skill will recognize that polynucleotides can bemanipulated in many ways and are not limited to the examples above.

A “polymerase polypeptide” of the present invention is a proteincomprising a polymerase domain. The polymerase polypeptide may alsocomprise additional domains including a heterologous DNA binding domain,e.g., Sso7D. DNA polymerases are well-known to those skilled in the art,including but not limited to DNA polymerases isolated or derived fromPyrococcus furiosus, Thermococcus litoralis, and Thermotoga maritime, ormodified versions there of. They include both DNA-dependent polymerasesand RNA-dependent polymerases such as reverse transcriptase. At leastfive families of DNA-dependent DNA polymerases are known, although mostfall into families A, B and C. There is little or no sequence similarityamong the various families. Most family A polymerases are single chainproteins that can contain multiple enzymatic functions includingpolymerase, 3′ to 5′ exonuclease activity and 5′ to 3′ exonucleaseactivity. Family B polymerases typically have a single catalytic domainwith polymerase and 3′ to 5′ exonuclease activity, as well as accessoryfactors. Family C polymerases are typically multi-subunit proteins withpolymerizing and 3′ to 5′ exonuclease activity. In E. coli, three typesof DNA polymerases have been found, DNA polymerases I (family A), II(family B), and III (family C). In eukaryotic cells, three differentfamily B polymerases, DNA polymerases a, 8, and c, are implicated innuclear replication, and a family A polymerase, polymerase y, is usedfor mitochondrial DNA replication. Other types of DNA polymerasesinclude phage polymerases. Similarly, RNA polymerases typically includeeukaryotic RNA polymerases I, II, and III, and bacterial RNA polymerasesas well as phage and viral polymerases. RNA polymerases can beDNA-dependent and RNA-dependent.

Polypeptide polymerases of the present invention have polymeraseactivity. Using the assays described herein, the activity of thepolypeptides of the present invention can be measured. Some polymerasepolypeptides of the invention exhibit improved polymerase activity ascompared to wild type polymerases in the assays described herein.

Two nucleic acid sequences or polypeptides are said to be “identical” ifthe sequence of nucleotides or amino acid residues, respectively, in thetwo sequences is the same when aligned for maximum correspondence asdescribed below. The terms “identical” or percent “identity,” in thecontext of two or more nucleic acids or polypeptide sequences, refer totwo or more sequences or subsequences that are the same or have aspecified percentage of amino acid residues or nucleotides that are thesame, when compared and aligned for maximum correspondence over acomparison window, as measured using one of the following sequencecomparison algorithms or by manual alignment and visual inspection. Whenpercentage of sequence identity is used in reference to proteins orpeptides, it is recognized that residue positions that are not identicaloften differ by conservative amino acid substitutions, where amino acidsresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. Where sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percentage sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated according to, e.g., the algorithm of Meyers& Miller, Computer Applic. Biol. Sci. 4:11-17 (1988) e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif., USA).

The term “substantial identity” of polynucleotide sequences means that apolynucleotide comprises a sequence that has at least 50% sequenceidentity. Exemplary embodiments include at least: 55%, 60, 65%, 70%,75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, or 99% compared to areference sequence using the programs described herein; preferably BLASTusing standard parameters, as described below. One of skill willrecognize that these values can be appropriately adjusted to determinecorresponding identity of proteins encoded by two nucleotide sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning and the like. Substantial identity of amino acidsequences for these purposes normally means sequence identity of atleast 65%. Exemplary embodiments include at least 65%, 70%, 75%, 80%,85%, 90%, 95% 94%, 95%, 96%, 97%, 98% or 99%. Polypeptides which are“substantially similar” share sequences as noted above except thatresidue positions which are not identical may differ by conservativeamino acid changes. Conservative amino acid substitutions refer to theinterchangeability of residues having similar side chains. For example,a group of amino acids having aliphatic side chains is glycine, alanine,valine, leucine, and isoleucine; a group of amino acids havingaliphatic-hydroxyl side chains is serine and threonine; a group of aminoacids having amide-containing side chains is asparagine and glutamine; agroup of amino acids having aromatic side chains is phenylalanine,tyrosine, and tryptophan; a group of amino acids having basic sidechains is lysine, arginine, and histidine; and a group of amino acidshaving sulfur-containing side chains is cysteine and methionine.Exemplary conservative amino acids substitution groups are:valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.

One of skill in the art will recognize that two polypeptides can also be“substantially identical” if the two polypeptides are immunologicallysimilar. Thus, overall protein structure may be similar while the aminoacid sequences of the two polypeptides show significant variation.Therefore, a method to measure whether two polypeptides aresubstantially identical involves measuring the binding of monoclonal orpolyclonal antibodies to each polypeptide. Two polypeptides aresubstantially identical if the antibodies specific for a firstpolypeptide bind to a second polypeptide with an affinity of at leastone third of the affinity for the first polypeptide. Two polypeptidesmay also be deemed to be “substantially identical” if they showcross-reactivity in a western blot analysis conducted using methodsknown in the art.

For sequence comparison, typically one sequence acts as a referencesequence, to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are entered into acomputer, subsequence coordinates are designated, if necessary, andsequence algorithm program parameters are designated. Default programparameters can be used, or alternative parameters can be designated. Thesequence comparison algorithm then calculates the percent sequenceidentities for the test sequences relative to the reference sequence,based on the program parameters.

A “comparison window”, as used herein, includes reference to a segmentof any one of the number of contiguous positions selected from the groupconsisting of from 20 to 600, usually about 50 to about 200, moreusually about 100 to about 150 in which a sequence may be compared to areference sequence of the same number of contiguous positions after thetwo sequences are optimally aligned. Methods of alignment of sequencesfor comparison are well-known in the art. Optimal alignment of sequencesfor comparison can be conducted, e.g., by the local homology algorithmof Smith & Waterman, Adv. Appl Math. 2:482 (1981), by the homologyalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, Proc. Nat'l.Acad. Sci. USA 85:2444 (1988), by computerized implementations of thesealgorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin GeneticsSoftware Package, Accelrys), or by manual alignment and visualinspection.

An example of algorithm that is suitable for determining percentsequence identity and sequence similarity is the BLAST algorithm, whichis described in Altschul et al., J. Mol. Biol. 215:403-410 (1990).Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information on the world wide web atncbi.nlm.nih.gov/). This algorithm involves first identifying highscoring sequence pairs (HSPs) by identifying short words of length W inthe query sequence, which either match or satisfy some positive-valuedthreshold score T when aligned with a word of the same length in adatabase sequence. T is referred to as the neighborhood word scorethreshold (Altschul et al, supra). These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are extended in both directions along each sequencefor as far as the cumulative alignment score can be increased. Extensionof the word hits in each direction are halted when: the cumulativealignment score falls off by the quantity X from its maximum achievedvalue; the cumulative score goes to zero or below, due to theaccumulation of one or more negative-scoring residue alignments; or theend of either sequence is reached. The BLAST algorithm parameters W, T,and X determine the sensitivity and speed of the alignment. The BLASTprogram uses as defaults a word length (W) of 11, the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915(1989)) alignments (B) of 50, expectation (E) of 10, M=5, N=−4, and acomparison of both strands.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin & Altschul, Proc.Nat'l. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The phrase “stringent hybridization conditions” refers to conditionsunder which a probe will hybridize to its target subsequence, typicallyin a complex mixture of nucleic acid, but to no other sequences.Stringent conditions are sequence-dependent and will be different indifferent circumstances. Longer sequences hybridize specifically athigher temperatures. An extensive guide to the hybridization of nucleicacids is found in Tijssen, Techniques in Biochemistry and MolecularBiology—Hybridization with Nucleic Probes, “Overview of principles ofhybridization and the strategy of nucleic acid assays” (1993).Generally, highly stringent conditions are selected to be about 5-10° C.lower than the thermal melting point (T_(m)) for the specific sequenceat a defined ionic strength pH. Low stringency conditions are generallyselected to be about 15-30° C. below the T_(m). The T_(m) is thetemperature (under defined ionic strength, pH, and nucleicconcentration) at which 50% of the probes complementary to the targethybridize to the target sequence at equilibrium (as the target sequencesare present in excess, at T_(m), 50% of the probes are occupied atequilibrium). Hybridization conditions are typically those in which thesalt concentration is less than about 1.0 M sodium ion, typically about0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3and the temperature is at least about 30° C. for short probes (e.g., 10to 50 nucleotides) and at least about 60° C. for long probes (e.g.,greater than 50 nucleotides). Stringent conditions may also be achievedwith the addition of destabilizing agents such as formamide. Forselective or specific hybridization, a positive signal is at least twotimes background, preferably 10 times background hybridization.

Nucleic acids that do not hybridize to each other under stringentconditions are still substantially identical if the polypeptides thatthey encode are substantially identical. This occurs, for example, whena copy of a nucleic acid is created using the maximum codon degeneracypermitted by the genetic code. In such cases, the nucleic acidstypically hybridize under moderately stringent hybridization conditions.Exemplary “moderately stringent hybridization conditions” include ahybridization in a buffer of 40% formamide, 1 M NaCl, 1% SDS at 37° C.,and a wash in 1×SSC at 45° C. Exemplary “stringent hybridizationconditions” include a hybridization in a buffer of 40% formamide, 1 MNaCl, 1% SDS at 37° C., and at least one wash in 0.2×SSC at atemperature of at least about 50° C., usually about 55° C. to about 60°C., for 20 minutes, or equivalent conditions. A positive hybridizationis at least twice background. Those of ordinary skill will readilyrecognize that alternative hybridization and wash conditions can beutilized to provide conditions of similar stringency.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Therecognized immunoglobulin genes include the kappa and lambda light chainconstant region genes, the alpha, gamma, delta, epsilon and mu heavychain constant region genes, and the myriad immunoglobulin variableregion genes. Antibodies exist, e.g., as intact immunoglobulins or as anumber of well characterized fragments produced by digestion withvarious peptidases. This includes, e.g., Fab′ and F(ab)′₂ fragments. Theterm “antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It also includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. The “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH1, CH2 and CH3, butdoes not include the heavy chain variable region.

The term “osmolyte” as used herein refers to any substance or compoundthat affects osmosis and/or contributes to the regulation of osmoticpressure in tissues and cells.

Overview

The present invention provides methods and compositions for improvingthe efficiency and specificity of nucleic acid amplification reactions.

In one aspect, the present invention provides a hybrid polymerasecomprising a polymerase domain and a DNA binding domain. In oneembodiment, hybrid polymerases of the invention comprise mutations thatrender the polymerases exonuclease deficient. “Exonuclease deficient” asused herein means that the polymerase has a substantially reduced (i.e.,less than 10%, 5% or 1% of the 3′-5′ exonuclease activity of Pfu DNApolymerase from Pyrococcus furiosus) or no exonuclease activity. Forexample, a double point mutation in the polymerase domain substitutingan alanine at positions D141 and E143 can remove or eliminate 3′-5′exonuclease activity. Derbyshire et al., Methods in Enzymology, Vol 262(1995), pages 363-385. Hybrid polymerases comprising such double pointmutations will generally show an increased specificity in nucleic acidamplification reactions, resulting in fewer amplification byproducts(such as amplification of primer-dimers) and increased efficiency inamplification of the desired target nucleic acids.

In a further embodiment, the DNA binding domain of hybrid polymerases ofthe invention comprises the Sso7d protein. Conjugation of polymerasedomains to DNA binding domains such as Sso7d increases the processivityof the polymerases, resulting in an increased amount of amplificationproduct.

In a further aspect, the invention provides methods, compositions,reaction mixtures, and kits for performing nucleic acid amplificationreactions that use hybrid polymerases complexed with hot-startantibodies. Such hot-start antibodies bind to the hybrid polymerases insuch a way as to inhibit polymerase activity at lower (ambient)temperatures. For example, in some embodiments, he antibodiessubstantially inhibit the polymerase activity at 50° C., 60° C., andeven 72° C., so that substantially no DNA synthesis occurs during thereaction setup at room temperature as well as during the initial heatingup of the thermal cycler block from room temperature to, e.g., 95° C.These antibodies will disassociate from the polymerases at highertemperatures, thus releasing the inhibition of the polymerase activity.Such antibodies increase the efficiency of nucleic acid amplificationreactions, because the polymerase remains inactive during initial set-upof the amplification reaction and prior to the initial denaturationstep. Since it is inactive at low temperatures, the polymerase complexedwith the antibody cannot elongate non-specific primer-template hybridsthat may form at the lower temperatures.

In a still further aspect, the invention provides methods, compositions,reaction mixtures and kits for performing nucleic acid amplificationreactions in the presence of additives that serve to improve theefficiency and specificity of such reactions. In one embodiment, thenucleic acid amplification reaction is conducted in the presence of anosmolyte, such as sarcosine. Sarcosine is known to stabilize proteins inaqueous solutions. Conventional methods of using sarcosine utilizeconcentrations in the molar range, but in the present invention,concentrations in the millimolar range, generally 50 millimolar orlower, resulted in improved efficiency of the amplification reaction. Ina further embodiment, molecules that mimic the electrostatic property ofdouble-stranded DNA are included in the reaction mixture to reducenon-specific binding of the polymerase to double-stranded templatenucleic acids. In a still further embodiment, the molecules included toreduce such non-specific binding are heparin molecules.

Hybrid Polymerases Useful in the Present Invention

The present invention provides hybrid polymerases comprising apolymerase domain and a DNA binding domain. Such hybrid polymerases areknown to show an increased processivity. See e.g., U.S. PatentApplication Publication Nos. 2006/005174; 2004/0219558;

2004/0214194; 2004/0191825; 2004/0081963; 2004/0002076; 2003/0162173;2003/0148330; 2003/0138830 and U.S. Pat. Nos. 6,627,424 and 7,445,898,each of which is hereby incorporated by reference in its entirety forall purposes and in particular for all teachings related to polymerases,hybrid/chimeric polymerases, as well as all methods for making and usingsuch polymerases.

In one aspect, the present invention provides hybrid polymerases thatlack 3′-5′ exonuclease activity. In one embodiment, such hybridpolymerases comprise a double point mutation in the polymerase domainthat provides this exonuclease deficiency. A variety of mutations can beintroduced into a native polymerase domain to reduce or eliminate 3′-5′exonuclease activity. For example, U.S. Pat. Nos. 6,015,668; 5,939,301and 5,948,614 describe mutations of a metal-binding aspartate to analanine residue in the 3′-5′ exonuclease domain of the Tma and Tne DNApolymerases. These mutations reduce the 3′-5′ exonuclease activities ofthese enzymes to below detectable levels. Similarly, U.S. Pat. No.5,882,904 describes an analogous aspartate-to-alanine mutation inThermococcus barossi, and U.S. Pat. No. 5,489,523 teaches thedouble-mutant D141A E143A of the Pyrococcus wosei DNA polymerases. Bothof these mutant polymerases have virtually no detectable 3′-5′exonuclease activity. Methods of assaying 3′-5′ exonuclease activity arewell-known in the art. See, e.g., Freemont et al., Proteins 1:66 (1986);Derbyshire et al., EMBO J. 16:17 (1991) and Derbyshire et al., Methodsin Enzymology 262:363 85 (1995). It will be understood that while theabove-described mutations were originally identified in one polymerase,one can generally introduce such mutations into other polymerases toreduce or eliminate exonuclease activity. In a specific embodiment, apolymerases of the invention comprise the double point mutationD141A/E143A in the polymerase domain. The phrase “corresponding to aposition,” in reference to polymerase amino acids, refers to an aminoacid that aligns with the same amino acid (e.g., D141 or E143) in areference polymerase amino acid sequence (e.g., SEQ ID NO:2). Sequencecomparisons can be performed using any BLAST including BLAST 2.2algorithm with default parameters, described in Altschul et al., Nuc.Acids Res. 25:3389 3402 (1977) and Altschul et al., J. Mol. Biol.215:403 410 (1990), respectively.

In a further embodiment, hybrid polymerases of the invention are encodedby a nucleotide sequence according to SEQ ID NO: 1. In a still furtherembodiment, hybrid polymerases of the invention are encoded by anucleotide sequence that has about 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequenceidentity to SEQ ID NO:1.

In a further embodiment, hybrid polymerases of the invention have anamino acid sequence according to SEQ ID NO: 2. In a still furtherembodiment, hybrid polymerases of the invention have an amino acidsequence with about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ IDNO: 2.

In some embodiments, the binding domain of hybrid polymerases of theinvention are from a thermostable organism and provides enhancedactivity at higher temperatures, e.g., temperatures above 45° C. Forexample, Sso7d and Sac7d are small (about 7 kd MW), basic chromosomalproteins from the hyperthermophilic archaeabacteria Sulfolobussolfataricus and S. acidocaldarius, respectively (see, e.g., Choli etal., Biochimica et Biophysica Acta 950:193-203, 1988; Baumann et al.,Structural Biol. 1:808-819, 1994; and Gao et al, Nature Struc. Biol.5:782-786, 1998). These proteins bind DNA in a sequence-independentmanner and when bound, increase the T_(m) of DNA by up to 40° C. undersome conditions (McAfee et al., Biochemistry 34:10063-10077, 1995).These proteins and their homologs are often used as thesequence-non-specific DNA binding domain in improved polymerase fusionproteins. Sso7d, Sac7d, Sac7e and related sequences (referred to hereinas “Sso7 sequences” or “Sso7 domains”) are known in the art (see, e.g.,accession numbers (P39476 (Sso7d); P13123 (Sac7d); and P13125 (Sac7e)).These sequences typically have at least 75% or greater, of 80%, 85%,90%, or 95% or greater, amino acid sequence identity. For example, anSso7 protein typically has at least 75% identity to an Sso7d sequence.

In further embodiments, hybrid polymerases of use in the presentinvention are described for example in U.S. Patent ApplicationPublication Nos. 2006/005174; 2004/0219558; 2004/0214194; 2004/0191825;2004/0081963; 2004/0002076; 2003/0162173; 2003/0148330; 2003/0138830 andU.S. Pat. Nos. 6,627,424 and 7,445,898, each of which is herebyincorporated by reference in its entirety for all purposes and inparticular for all teachings related to polymerases, hybrid/chimericpolymerases, as well as all methods for making and using suchpolymerases. Examples of hybrid polymerase proteins and methods ofgenerating hybrid proteins are also disclosed in WO2004011605, which ishereby incorporated by reference in its entirety for all purposes, andin particular for all teachings related to generating hybrid proteins.

One advantage of some of the polymerases of the invention is theirability to amplify target nucleic acids in the presence of a substanceat concentrations of the substance that typically inhibits PCRamplifications using wildtype Taq polymerase. Examples of suchinhibitory substances include, but are not limited to, blood, intactcells, and food samples. Accordingly, the polymerases of the inventioncan be used to amplify nucleic acids in whole blood or serum, optionallywhere the blood or serum are not purified before addition to thereaction mixture. In some embodiments, the polymerases of the inventionare used to amplify whole cells, including but not limited to wholebacterial, fungal, or yeast cells, e.g., in whole colony PCR. In someembodiments, the polymerases of the invention are used to amplify anucleic acid in a food sample, optionally without further purificationor with partial purification of the nucleic acid from the food sampleprior to amplification. This is useful, for example, for screening forbacterial, fungal, or viral contamination of foodstuffs. Exemplary foodsamples include, but are not limited to, samples comprising coco,cheese, meat, egg, etc.

In some embodiments, the polymerases of the invention are capable ofparticularly rapid amplification due to its improved processivity. Forexample, in some embodiments the polymerases of the invention are usedin an amplification reaction having an extension time of less than 5, 4,3, 2, or 1 second, and yet amplifying the target nucleic acid, e.g.,with high efficiency (e.g., greater than 90%, 95% or more).

Polymerase Domains

In one exemplary embodiment, hybrid polymerases of the invention have apolymerase domain derived from two parental polymerases, Pfu andDeepVent. Such polymerases are described for example in U.S. ApplicationPublication Nos. 20040219558; 20040214194; 20040191825; 20030162173,each of which is hereby incorporated by reference in its entirety forall purposes and in particular for all teachings related to hybridpolymerases.

A variety of polymerases can be used as at least a portion of thepolymerase domain of hybrid polymerases of the invention. At least fivefamilies of DNA-dependent DNA polymerases are known, although most fallinto families A, B and C. There is little or no structural or sequencesimilarity among the various families. Most family A polymerases aresingle chain proteins that can contain multiple enzymatic functionsincluding polymerase, 3′ to 5′ exonuclease activity and 5′ to 3′exonuclease activity. Family B polymerases typically have a singlecatalytic domain with polymerase and 3′ to 5′ exonuclease activity, aswell as accessory factors. Family C polymerases are typicallymulti-subunit proteins with polymerizing and 3′ to 5′ exonucleaseactivity. In E. coli, three types of DNA polymerases have been found,DNA polymerases I (family A), II (family B), and III (family C). Ineukaryotic cells, three different family B polymerases, DNA polymerasesa, 6, and c are implicated in nuclear replication, and a family Apolymerase, polymerase y, is used for mitochondrial DNA replication.Other types of DNA polymerases include phage polymerases. Any of thesepolymerases, combinations of all or portions of these polymerases, aswell as chimeras or hybrids between two or more of such polymerases ortheir equivalents can be used to form a portion or all of the polymerasedomain of hybrid polymerases of the invention.

Further, in some embodiments, non-thermostable polymerases may also beused in accordance with the invention. For example, the large fragmentof E. coli DNA Polymerase I (Klenow) (the Klenow Fragment) with mutation(D355A, E357A) abolishes the 3′=>5′ exonuclease activity. This enzyme orequivalent enzymes can be used in embodiments where the amplificationreaction is not performed at high temperatures.

In some embodiments, the hybrid polymerases of the invention include apolymerase domain comprising mutations that reduce or abolishexonuclease activity of any hybrid polymerase comprising such apolymerase domain in comparison to a hybrid polymerase comprising apolymerase domain that does not have such mutations. In furtherembodiments, such hybrid polymerases comprise mutations in theexonuclease domain. In still further embodiments, such hybridpolymerases comprise an amino acid sequence according to SEQ ID NO: 2.

Nucleic Acid Binding Domains

In some embodiments, hybrid polymerases of the invention comprise apolymerase domain conjugated to a DNA binding domain. A DNA bindingdomain is a protein, or a defined region of a protein, that binds tonucleic acid in a sequence-independent matter, e.g., binding does notexhibit a gross preference for a particular sequence. DNA bindingdomains may bind single or double stranded nucleic acids.

The DNA binding proteins of use in the invention are generallythermostable. Examples of such proteins include, but are not limited to,the Archaeal small basic DNA binding proteins Sso7d and Sso7d-likeproteins (see, e.g., Choli et al., Biochimica et Biophysica Acta950:193-203, 1988; Baumann et al., Structural Biol. 1:808-819, 1994; andGao et al, Nature Struc. Biol. 5:782-786, 1998), Archaeal HMf-likeproteins (see, e.g., Stanch et al., J. Molec. Biol. 255:187-203, 1996;Sandman et al., Gene 150:207-208, 1994), and PCNA homologs (see, e.g.,Cann et al., J. Bacteriology 181:6591-6599, 1999; Shamoo and Steitz,Cell: 99, 155-166, 1999; De Felice et al., J. Molec. Biol. 291, 47-57,1999; and Zhang et al., Biochemistry 34:10703-10712, 1995).

Sso7d and Sso7d-like proteins, Sac7d and Sac7d-like proteins, e.g.,Sac7a, Sac7b, Sac7d, and Sac7e are small (about 7,000 kd MW), basicchromosomal proteins from the hyperthermophilic archaebacteriaSulfolobus solfataricus and S. acidocaldarius, respectively. Theseproteins are lysine-rich and have high thermal, acid and chemicalstability. They bind DNA in a sequence-independent manner and whenbound, increase the T_(m) of DNA by up to 40° C. under some conditions(McAfee, Biochemistry 34:10063-10077, 1995; Gao et al., Nat. Struct.Biol. 5(9):782-786, 1998). These proteins and their homologs aretypically believed to be involved in stabilizing genomic DNA at elevatedtemperatures. Suitable Sso7d-like DNA binding domains for use in theinvention can be modified based on their sequence homology to Sso7d.Typically, DNA binding domains that are identical to or substantiallyidentical to a known DNA binding protein over a comparison window ofabout 25 amino acids, optionally about 50-100 amino acids, or the lengthof the entire protein, can be used in the invention. The sequence can becompared and aligned for maximum correspondence over a comparisonwindow, or designated region as measured using one of the describedcomparison algorithms or by manual alignment and visual inspection. Insome embodiments, the DNA polymerase comprises SEQ ID NO: 3 or asubstantially (e.g., at least 60%, 70%, 80%, 90%, 95%) identicalsequence thereof. A variety of mutations in the Sso7 binding domain havebeen described in, e.g., US Patent Application Nos. 2005/0048530 and2007/0141591.

The HMf-like proteins are archaeal histones that share homology both inamino acid sequences and in structure with eukaryotic H4 histones, whichare thought to interact directly with DNA. The HMf family of proteinsform stable dimers in solution, and several HMf homologs have beenidentified from thermostable species (e.g., Methanothermus fervidus andPyrococcus strain GB-3a). The HMf family of proteins, once joined to TaqDNA polymerase or any DNA modifying enzyme with a low intrinsicprocessivity, can enhance the ability of the enzyme to slide along theDNA substrate and thus increase its processivity. For example, thedimeric HMf-like protein can be covalently linked to the N terminus ofTaq DNA polymerase, e.g., via chemical modification, and thus improvethe processivity of the polymerase.

Certain helix-hairpin-helix motifs have been shown to bind DNAnonspecifically and enhance the processivity of a DNA polymerase towhich it is fused (Pavlov et al., Proc Natl Acad Sci USA. 99:13510-5,2002).

Additional DNA binding domains suitable for use in the invention can beidentified by homology with known DNA binding proteins and/or byantibody crossreactivity, or may be found by means of a biochemicalassay. DNA binding domains may be synthesized or isolated using thetechniques described herein and known in the art.

Sequence non-specific doubled-stranded nucleic acid binding domains foruse in the invention can also be identified by cross-reactivity usingantibodies, including but not limited to polyclonal antibodies, thatbind to known nucleic acid binding domains. Polyclonal antibodies aregenerated using methods well known to those of ordinary skill in the art(see, e.g., Coligan, Current Protocols in Immunology (1991); Harlow &Lane, Antibodies, A Laboratory Manual (1988)). Those proteins that areimmunologically cross-reactive binding proteins can then be detected bya variety of assay methods. For descriptions of various formats andconditions that can be used, see, e.g., Methods in Cell Biology:Antibodies in Cell Biology, volume 37 (Asai, ed. 1993), Coligan, supra,and Harlow & Lane, supra.

Specificity for binding to double-stranded nucleic acids can be testedusing a variety of assays known to those of ordinary skill in the art.These include such assays as filter binding assays or gel-shift assays.For example, in a filter-binding assay the polypeptide to be assessedfor binding activity to double-stranded DNA is pre-mixed withradio-labeled DNA, either double-stranded or single-stranded, in theappropriate buffer. The mixture is filtered through a membrane (e.g.,nitrocellulose) which retains the protein and the protein-DNA complex.The amount of DNA that is retained on the filter is indicative of thequantity that bound to the protein. Binding can be quantified by acompetition analysis in which binding of labeled DNA is competed by theaddition of increasing amounts of unlabelled DNA. A polypeptide thatbinds double-stranded DNA at a 10-fold or greater affinity thansingle-stranded DNA is defined herein as a double-stranded DNA bindingprotein. Alternatively, binding activity can be assessed by a gel shiftassay in which radiolabeled DNA is incubated with the test polypeptide.The protein-DNA complex will migrate slower through the gel than unboundDNA, resulting in a shifted band. The amount of binding is assessed byincubating samples with increasing amounts of double-stranded orsingle-stranded unlabeled DNA, and quantifying the amount ofradioactivity in the shifted band.

A binding domain suitable for use in the invention binds todouble-stranded nucleic acids in a sequence-independent fashion, i.e., abinding domain of the invention binds double-stranded nucleic acids witha significant affinity, but, there is no known nucleic acid that bindsto the domain with more than 100-fold more affinity than another nucleicacid with the same nucleotide composition, but a different nucleic acidsequence. Non-specific binding can be assayed using methodology similarto that described for determining double-stranded vs. single-strandednucleic acid binding. Filter binding assays or gel mobility shift assayscan be performed as above using competitor DNAs of the same nucleotidecomposition, but different nucleic acid sequences to determinespecificity of binding.

Sequence non-specific double-stranded nucleic acid binding domains foruse in the invention can also be assessed, for example, by assaying theability of the double-stranded binding domain to increase processivityor efficiency of a modifying enzyme or to increase the stability of anucleic acid duplex by at least 1° C. can be determined.

A binding domain of the invention can also be identified by directassessment of the ability of such a domain to stabilize adouble-stranded nucleic acid conformation. For example, a melting curveof a primer-template construct can be obtained in the presence orabsence of protein by monitoring the UV absorbance of the DNA at 260 nm.The T_(m) of the double-stranded substrate can be determined from themidpoint of the melting curve. The effect of the sequence-non-specificdouble-stranded nucleic-acid-binding protein on the T_(m) can then bedetermined by comparing the T_(m) obtained in the presence of themodified enzyme with that in the presence of the unmodified enzyme. (Theprotein does not significantly contribute to the UV absorbance becauseit has a much lower extinction coefficient at 260 nm than DNA). A domainthat increases the T_(m) by 1° C., often by 5° C., 10° C. or more, canthen be selected for use in the invention.

Novel sequence non-specific double-stranded nucleic acid bindingproteins of the invention can also be isolated by taking advantage oftheir DNA binding activity, for instance by purification onDNA-cellulose columns. The isolated proteins can then be furtherpurified by conventional means, sequenced, and the genes cloned byconventional means via PCR. Proteins overexpressed from these clones canthen be tested by any of the means described above.

Producing Polymerases of the Invention

polymerases of the invention are generally produced by joining apolymerase domain to a DNA binding domain using chemical and/orrecombinant methods.

polymerases of the invention can be produced using techniques known inthe art. Methods for producing polymerases comprising a polymerasedomain and a nucleic acid binding domain are described, for example, inU.S. Patent Application Publication Nos. 2006/005174; 2004/0219558;2004/0214194; 2004/0191825; 2004/0081963; 2004/0002076; 2003/0162173;2003/0148330; 2003/0138830 and U.S. Pat. Nos. 6,627,424 and 7,445,898,each of which is hereby incorporated by reference in its entirety forall purposes and in particular for all teachings related to polymerases,hybrid/chimeric polymerases, as well as all methods for making and usingsuch polymerases.

Chemical methods of joining a DNA binding protein to a polymerase domainare described, e.g., in Bioconjugate Techniques, Hermanson, Ed.,Academic Press (1996). These include, for example, derivitization forthe purpose of linking the two proteins to each other, either directlyor through a linking compound, by methods that are well known in the artof protein chemistry. For example, in one chemical conjugationembodiment, the means of linking the catalytic domain and the DNAbinding domain comprises a heterobifunctional-coupling reagent whichultimately contributes to formation of an intermolecular disulfide bondbetween the two moieties. Other types of coupling reagents that areuseful in this capacity for the present invention are described, forexample, in U.S. Pat. No. 4,545,985. Alternatively, an intermoleculardisulfide may conveniently be formed between cysteines in each moiety,which occur naturally or are inserted by genetic engineering. The meansof linking moieties may also use thioether linkages betweenheterobifunctional crosslinking reagents or specific low pH cleavablecrosslinkers or specific protease cleavable linkers or other cleavableor noncleavable chemical linkages.

The methods of linking a DNA binding domain, e.g., Sso7d, and apolymerase domain may also comprise a peptidyl bond formed betweenmoieties that are separately synthesized by standard peptide synthesischemistry or recombinant means. The conjugate protein itself can also beproduced using chemical methods to synthesize an amino acid sequence inwhole or in part. For example, peptides can be synthesized by solidphase techniques, such as, e.g., the Merrifield solid phase synthesismethod, in which amino acids are sequentially added to a growing chainof amino acids (see, Merrifield (1963) J. Am. Chem. Soc., 85:2149-2146).Equipment for automated synthesis of polypeptides is commerciallyavailable from suppliers such as PE Corp. (Foster City, Calif.), and maygenerally be operated according to the manufacturer's instructions. Thesynthesized peptides can then be cleaved from the resin, and purified,e.g., by preparative high performance liquid chromatography (seeCreighton, Proteins Structures and Molecular Principles, 50-60 (1983)).The composition of the synthetic polypeptides or of subfragments of thepolypeptide, may be confirmed by amino acid analysis or sequencing(e.g., the Edman degradation procedure; see Creighton, Proteins,Structures and Molecular Principles, pp. 34-49 (1983)).

In another embodiment, a DNA binding domain and polymerase domain can bejoined via a linking group. The linking group can be a chemicalcrosslinking agent, including, for example,succinimidyl-(N-maleimidometh-yl)-cyclohexane-1-carboxylate (SMCC). Thelinking group can also be an additional amino acid sequence(s),including, for example, a polyalanine, polyglycine or similarly, linkinggroup.

In some embodiments, the coding sequences of each polypeptide in aresulting fusion protein (also referred to herein as “hybrid” and/or“chimeric” or “chimera” protein) are directly joined at their amino- orcarboxy-terminus via a peptide bond in any order. Alternatively, anamino acid linker sequence may be employed to separate the first andsecond polypeptide components by a distance sufficient to ensure thateach polypeptide folds into its secondary and tertiary structures. Suchan amino acid linker sequence is incorporated into the fusion proteinusing standard techniques well known in the art. Suitable peptide linkersequences may be chosen based on the following factors: (1) theirability to adopt a flexible extended conformation; (2) their inabilityto adopt a secondary structure that could interact with functionalepitopes on the first and second polypeptides; and (3) the lack ofhydrophobic or charged residues that might react with the polypeptidefunctional epitopes. Typical peptide linker sequences contain Gly, Ser,Val and Thr residues. Other near neutral amino acids, such as Ala canalso be used in the linker sequence. Amino acid sequences which may beusefully employed as linkers include those disclosed in Maratea et al.(1985) Gene 40:39-46; Murphy et al. (1986) Proc. Natl. Acad. Sci. USA83:8258-8262; U.S. Pat. Nos. 4,935,233 and 4,751,180, each of which ishereby incorporated by reference in its entirety for all purposes and inparticular for all teachings related to linkers. The linker sequence maygenerally be from 1 to about 50 amino acids in length, e.g., 3, 4, 6, or10 amino acids in length, but can be 100 or 200 amino acids in length.Linker sequences may not be required when the first and secondpolypeptides have non-essential N-terminal amino acid regions that canbe used to separate the functional domains and prevent stericinterference. In some embodiments, linker sequences of use in thepresent invention comprise an amino acid sequence according to SEQ IDNO: 5.

Other chemical linkers include carbohydrate linkers, lipid linkers,fatty acid linkers, polyether linkers, e.g., PEG, etc. For example,poly(ethylene glycol) linkers are available from Shearwater Polymers,Inc. Huntsville, Ala. These linkers optionally have amide linkages,sulfhydryl linkages, or heterobifunctional linkages.

Other methods of joining a DNA binding domain and polymerase domaininclude ionic binding by expressing negative and positive tails andindirect binding through antibodies and streptavidin-biotininteractions. (See, e.g., Bioconjugate Techniques, supra). The domainsmay also be joined together through an intermediate interactingsequence. For example, DNA binding domain-interacting sequence, i.e., asequence that binds to a particular DNA binding domain (such as Sso7d),can be joined to a polymerase. The resulting fusion protein can then beallowed to associate non-covalently with the DNA binding domain togenerate a DNA-binding-domain-polymerase conjugate.

As previously described, nucleic acids encoding the polymerase or DNAbinding domains can be obtained using routine techniques in the field ofrecombinant genetics. Basic texts disclosing the general methods of usein this invention include Sambrook and Russell, Molecular Cloning, ALaboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer andExpression: A Laboratory Manual (1990); and Current Protocols inMolecular Biology (Ausubel et al., eds., 1994-1999). Such nucleic acidsmay also be obtained through in vitro amplification methods such asthose described herein and in Berger, Sambrook, and Ausubel, as well asMullis et al., (1987) U.S. Pat. No. 4,683,202; PCR Protocols A Guide toMethods and Applications (Innis et al., eds) Academic Press Inc. SanDiego, Calif. (1990) (Innis); Arnheim & Levinson (Oct. 1, 1990) C&EN36-47; The Journal Of NIH Research (1991) 3: 81-94; Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl.Acad. Sci. USA 87, 1874; Lomell et al. (1989) J. Clin. Chem., 35: 1826;Landegren et al., (1988) Science 241: 1077-1080; Van Brunt (1990)Biotechnology 8: 291-294; Wu and Wallace (1989) Gene 4: 560; andBarringer et al. (1990) Gene 89: 117, each of which is incorporated byreference in its entirety for all purposes and in particular for allteachings related to amplification methods.

One of skill will recognize that modifications can additionally be madeto the polymerases of the present invention without diminishing theirbiological activity. Some modifications may be made to facilitate thecloning, expression, or incorporation of a domain into a fusion protein.Such modifications are well known to those of skill in the art andinclude, for example, the addition of codons at either terminus of thepolynucleotide that encodes the binding domain to provide, for example,a methionine added at the amino terminus to provide an initiation site,or additional amino acids (e.g., poly His) placed on either terminus tocreate conveniently located restriction sites or termination codons orpurification sequences.

The polymerases of the present invention can be expressed in a varietyof host cells, including E. coli, other bacterial hosts, yeasts,filamentous fungi, and various higher eukaryotic cells such as the COS,CHO and HeLa cells lines and myeloma cell lines. Techniques for geneexpression in microorganisms are described in, for example, Smith, GeneExpression in Recombinant Microorganisms (Bioprocess Technology, Vol.22), Marcel Dekker, 1994. Examples of bacteria that are useful forexpression include, but are not limited to, Escherichia, Enterobacter,Azotobacter, Erwinia, Bacillus, Pseudomonas, Klebsielia, Proteus,Salmonella, Serratia, Shigella, Rhizobia, Vitreoscilla, and Paracoccus.Filamentous fungi that are useful as expression hosts include, forexample, the following genera: Aspergillus, Trichoderma, Neurospora,Penicillium, Cephalosporium, Achlya, Podospora, Mucor, Cochliobolus, andPyricularia. See, e.g., U.S. Pat. No. 5,679,543 and Stahl and Tudzynski,Eds., Molecular Biology in Filamentous Fungi, John Wiley & Sons, 1992.Synthesis of heterologous proteins in yeast is well known and describedin the literature. Methods in Yeast Genetics, Sherman, F., et al., ColdSpring Harbor Laboratory, (1982) is a well recognized work describingthe various methods available to produce the enzymes in yeast.

There are many expression systems for producing the polymerasepolypeptides of the present invention that are well known to those ofordinary skill in the art. (See, e.g., Gene Expression Systems,Fernandex and Hoeffler, Eds. Academic Press, 1999; Sambrook and Russell,supra; and Ausubel et al, supra.) Typically, the polynucleotide thatencodes the variant polypeptide is placed under the control of apromoter that is functional in the desired host cell. Many differentpromoters are available and known to one of skill in the art, and can beused in the expression vectors of the invention, depending on theparticular application. Ordinarily, the promoter selected depends uponthe cell in which the promoter is to be active. Other expression controlsequences such as ribosome binding sites, transcription terminationsites and the like are also optionally included. Constructs that includeone or more of these control sequences are termed “expressioncassettes.” Accordingly, the nucleic acids that encode the joinedpolypeptides are incorporated for high level expression in a desiredhost cell.

Expression control sequences that are suitable for use in a particularhost cell are often obtained by cloning a gene that is expressed in thatcell. Commonly used prokaryotic control sequences, which are definedherein to include promoters for transcription initiation, optionallywith an operator, along with ribosome binding site sequences, includesuch commonly used promoters as the beta-lactamase (penicillinase) andlactose (lac) promoter systems (Change et al., Nature (1977) 198: 1056),the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res.(1980) 8: 4057), the tac promoter (DeBoer, et al., Proc. Natl. Acad.Sci. U.S.A. (1983) 80:21-25); and the lambda-derived PL promoter andN-gene ribosome binding site (Shimatake et al., Nature (1981) 292: 128).The particular promoter system is not critical to the invention, anyavailable promoter that functions in prokaryotes can be used. Standardbacterial expression vectors include plasmids such as pBR322-basedplasmids, e.g., pBLUESCRIPT™, pSKF, pET23D, lambda-phage derivedvectors, and fusion expression systems such as GST and LacZ. Epitopetags can also be added to recombinant proteins to provide convenientmethods of isolation, e.g., c-myc, HA-tag, 6-His tag (SEQ ID NO:46),maltose binding protein, VSV-G tag, anti-DYKDDDDK tag (SEQ ID NO:47), orany such tag, a large number of which are well known to those of skillin the art.

For expression in prokaryotic cells other than E. coli, a promoter thatfunctions in the particular prokaryotic species is required. Suchpromoters can be obtained from genes that have been cloned from thespecies, or heterologous promoters can be used. For example, the hybridtrp-lac promoter functions in Bacillus sp. in addition to E. coli. Theseand other suitable bacterial promoters are well known in the art and aredescribed, e.g., in Sambrook et al. and Ausubel et al. Bacterialexpression systems for expressing the proteins of the invention areavailable in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al.,Gene 22:229-235 (1983); Mosbach et al., Nature 302:543-545 (1983). Kitsfor such expression systems are commercially available.

Eukaryotic expression systems for mammalian cells, yeast, and insectcells are well known in the art and are also commercially available. Inyeast, vectors include Yeast Integrating plasmids (e.g., YIp5) and YeastReplicating plasmids (the YRp series plasmids) and pGPD-2. Expressionvectors containing regulatory elements from eukaryotic viruses aretypically used in eukaryotic expression vectors, e.g., SV40 vectors,papilloma virus vectors, and vectors derived from Epstein-Barr virus.Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+,pMAMneo-5, baculovirus pDSVE, and any other vector allowing expressionof proteins under the direction of the CMV promoter, SV40 earlypromoter, SV40 later promoter, metallothionein promoter, murine mammarytumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter,or other promoters shown effective for expression in eukaryotic cells.

Either constitutive or regulated promoters can be used in the presentinvention. Regulated promoters can be advantageous because the hostcells can be grown to high densities before expression of the fusionpolypeptides is induced. High level expression of heterologous proteinsslows cell growth in some situations. An inducible promoter is apromoter that directs expression of a gene where the level of expressionis alterable by environmental or developmental factors such as, forexample, temperature, pH, anaerobic or aerobic conditions, light,transcription factors and chemicals.

For E. coli and other bacterial host cells, inducible promoters areknown to those of skill in the art. These include, for example, the lacpromoter, the bacteriophage lambda PL promoter, the hybrid trp-lacpromoter (Amann et al. (1983) Gene 25: 167; de Boer et al. (1983) Proc.Nat'l. Acad. Sci. USA 80: 21), and the bacteriophage T7 promoter(Studier et al. (1986) J. Mol. Biol.; Tabor et al. (1985) Proc. Nat'lAcad. Sci. USA 82: 1074-8). These promoters and their use are alsodiscussed in Sambrook et al., supra.

Translational coupling may be used to enhance expression. The strategyuses a short upstream open reading frame derived from a highly expressedgene native to the translational system, which is placed downstream ofthe promoter, and a ribosome binding site followed after a few aminoacid codons by a termination codon. Just prior to the termination codonis a second ribosome binding site, and following the termination codonis a start codon for the initiation of translation. The system dissolvessecondary structure in the RNA, allowing for the efficient initiation oftranslation. See Squires, et. al. (1988), J. Biol. Chem. 263:16297-16302.

The construction of polynucleotide constructs generally requires the useof vectors able to replicate in bacteria. Such vectors are commonly usedin the art. A plethora of kits are commercially available for thepurification of plasmids from bacteria (for example, EasyPrep™,FlexiPrep™, from Pharmacia Biotech; StrataClean™, from Stratagene; and,QlAexpress® Expression System, Qiagen). The isolated and purifiedplasmids can then be further manipulated to produce other plasmids, andused to transform cells.

The polypeptides of the present invention can be expressedintracellularly, or can be secreted from the cell. Intracellularexpression often results in high yields. If necessary, the amount ofsoluble, active fusion polypeptide may be increased by performingrefolding procedures (see, e.g., Sambrook et al., supra.; Marston etal., Bio/Technology (1984) 2: 800; Schoner et al., Bio/Technology (1985)3: 151). Polypeptides of the invention can be expressed in a variety ofhost cells, including E. coli, other bacterial hosts, yeast, and varioushigher eukaryotic cells such as the COS, CHO and HeLa cells lines andmyeloma cell lines. The host cells can be mammalian cells, insect cells,or microorganisms, such as, for example, yeast cells, bacterial cells,or fungal cells.

Once expressed, the polypeptides can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, gel electrophoresis and thelike (see, generally, R. Scopes, Protein Purification, Springer-Verlag,N.Y. (1982), Deutscher, Methods in Enzymology Vol. 182: Guide to ProteinPurification., Academic Press, Inc. N.Y. (1990)). Substantially purecompositions of at least about 90 to 95% homogeneity are preferred, and98 to 99% or more homogeneity are most preferred. Once purified,partially or to homogeneity as desired, the polypeptides may then beused (e.g., as immunogens for antibody production).

To facilitate purification of the polypeptides of the invention, thenucleic acids that encode the polypeptides can also include a codingsequence for an epitope or “tag” for which an affinity binding reagentis available. Examples of suitable epitopes include the myc and V-5reporter genes; expression vectors useful for recombinant production offusion polypeptides having these epitopes are commercially available(e.g., Invitrogen (Carlsbad Calif.) vectors pcDNA3.1/Myc-His andpcDNA3.1/V5-His are suitable for expression in mammalian cells).Additional expression vectors suitable for attaching a tag to the fusionproteins of the invention, and corresponding detection systems are knownto those of skill in the art, and several are commercially available(e.g., FLAG″ (Kodak, Rochester N.Y.). Another example of a suitable tagis a polyhistidine sequence, which is capable of binding to metalchelate affinity ligands. Typically, six adjacent histidines (SEQ IDNO:46) are used, although one can use more or less than six. Suitablemetal chelate affinity ligands that can serve as the binding moiety fora polyhistidine tag include nitrilo-tri-acetic acid (NTA) (Hochuli, E.(1990) “Purification of recombinant proteins with metal chelatingadsorbents” In Genetic Engineering: Principles and Methods, J. K.Setlow, Ed., Plenum Press, N.Y.; commercially available from Qiagen(Santa Clarita, Calif.)).

One of skill in the art would recognize that after biological expressionor purification, the polymerase peptide (s) may possess a conformationsubstantially different than the native conformations of the constituentpolypeptides. In this case, it may be necessary or desirable to denatureand reduce the polypeptide and then to cause the polypeptide to re-foldinto the preferred conformation. Methods of reducing and denaturingproteins and inducing re-folding are well known to those of skill in theart (See, Debinski et al. (1993) J. Biol. Chem. 268: 14065-14070;Kreitman and Pastan (1993) Bioconjug. Chem. 4: 581-585; and Buchner etal. (1992) Anal. Biochem. 205: 263-270). Debinski et al., for example,describe the denaturation and reduction of inclusion body proteins inguanidine-DTE. The protein is then refolded in a redox buffer containingoxidized glutathione and L-arginine.

Hot-Start Methods

In some embodiments, hybrid polymerases of the invention are used innucleic acid amplification methods, particularly quantitative PCR (qPCR)methods. In such amplification methods, it can be beneficial to employ“hot start” methods to decrease the generation of primer dimers andunspecific amplification products at ambient temperatures. A number ofhot-start methods are known. These include physical separation of thepolymerase, use of nucleic acid additives to inhibit extension reactionsat low temperatures, and modifications to the active site of thepolymerase. Often, it may be desirable to use “hot start” polymerases.In a hot-start polymerase, a molecule is typically bound to the enzymeat the active site to inhibit polymerase activity at lower temperatures.The molecule is removed at high temperatures (e.g., at 95° C.) to allowthe polymerase to function at the desired point of the process. Themolecule can be one or more antibody, peptide, or a small organicmolecule. For example, hot-start can be achieved using one or moreantibody that binds to a polymerase with high affinity at ambienttemperatures in an inhibitory manner. The complex is dissociated in ahigh temperature preheating step.

A polymerase may also be chemically modified for hot-start. Heat labileblocking groups are introduced into the polymerase, which render theenzyme inactive at room temperature. These blocking groups are removedat high temperature prior to cycling such that the enzyme is activated.Heat labile modifications include coupling citraconic anhydride oraconitric anhydride to lysine residues of the enzyme are known in theart, see e.g., U.S. Pat. No. 5,677,152, which is hereby incorporated byreference in its entirety for all purposes and in particular for allteachings related to hot-start methods.

U.S. Patent Application Publication No. 2003/0119150 also discloses aconcept of hot start PCR that employs a thermostable exonuclease and apolymerase. This method is based on preventing primer elongation at lowtemperatures by introducing chemical modifications at the 3′ end of atleast one primer. A thermostable exonuclease is used that is inactive atambient temperatures or below. Upon temperature increase, theexonuclease becomes active and capable of removing the 3′ modificationof the primer to enable it to participate in the amplification reaction.U.S. 20030119150, which is hereby incorporated by reference in itsentirety for all purposes and in particular for all teachings related tohot-start methods, further teaches that when hybridization probes areused for real-time monitoring, e.g., TaqMan hybridization probes,Molecular Beacon oligonucleotides, or two oligonucleotide hybridizationmethods, the presence of a thermostable exonuclease III requires asuitable blocking method for the 3′ end of the detection probe to avoid3′ digestion.

Hot-Start Antibodies

In certain embodiments of the invention, monoclonal antibodies are usedto provide the hot-start features to hybrid polymerases of theinvention.

In one aspect, the present invention provides methods for producing andscreening for the appropriate antibodies against hybrid polymerases ofthe invention, particularly those polymerases that exhibit both 5′ to 3′polymerase activity and 3′ to 5′ exonuclease activity. For suchpolymerases, it can be beneficial to use one or more antibodies that cansufficiently inhibit either or both the polymerase and the exonucleaseactivity of the hybrid polymerase. Important features of antibodies ofuse in this aspect of the invention include binding affinity for thepolymerase as well as the ability to block polymerase and/or exonucleaseactivities.

Hot-start antibodies increase the specificity of amplificationreactions, because they render the polymerase inactive at roomtemperature, thus avoiding extension of nonspecifically annealed primersor primer dimers. The functional activity of the polymerase is restoredby disassociating the antibody from the polymerase, generally throughincubation at a higher temperature. In some embodiment, such a “highertemperature” is from about 90° to about 99° C. for about 2 to about 10minutes. It will be appreciated that the temperature and length of timefor incubation to disassociate the antibody and activate the polymerasecan be varied according to known parameters to provide the mosteffective method of activating the polymerase in these hot-startmethods.

Methods for screening for antibodies of use in the present inventioninclude methods known in the art, such as affinity-based ELISA assays,as well as functional assays for polymerase and/or exonucleaseinhibition. For such functional assays, the amount of DNA produced ordigested per unit of time can be correlated to the activity of thepolymerase or exonuclease used, thus providing an estimate of the amountof inhibition a particular antibody can exert on either or both thepolymerase and exonuclease activity of the polymerase.

In one aspect, the present invention provides antibodies that bind to apolymerase including but not limited to a polymerase comprising SEQ IDNO:2. In some embodiments, the antibodies inhibit DNA polymeraseactivity, and/or, when present 3′-5′ exonuclease activity. In someembodiments, the antibodies inhibit DNA polymerase and/or 3′-5′exonuclease activity of a polymerase comprising SEQ ID NO:2 but whereposition 141 is D and 143 is E in cases where exonuclease activity ismeasured, but does not significantly inhibit the same polymeraseactivity of Taq polymerase. In some embodiments, the antibodies inhibitthe DNA polymerase and/or exonuclease activity of a DNA polymerase by atleast 80% or 90% but does not inhibit DNA polymerase activity of Taqpolymerase by more than 15%. Four exemplary antibodies are providedherein as well as sequence information related to their heavy and lightvariable regions, including all CDR sequences. In some embodiments, theantibodies described herein are complexed with a polymerase.

In some exemplary embodiments, hot-start antibodies of use in thepresent invention comprise light-chain variable regions with nucleotidesequences of about 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to any one of SEQ ID NOs: 10-13. Such monoclonalantibodies may in further exemplary embodiments comprise heavy-chainvariable regions with nucleotide sequences of about 75%, 80%, 85%, 90%,94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQID NOs: 6-9. Such monoclonal antibodies may in still further exemplaryembodiments comprise heavy-chain variable regions with amino acidsequences of about 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or100% sequence identity to any one of SEQ ID NOs: 14-17 and/orlight-chain variable regions with amino acid sequences of about 75%,80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toany one of SEQ ID NOs: 18-21.

In some embodiments, an antibody is provided comprising a heavy chainvariable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:22, SEQ ID NO:23, and SEQ ID NO:24, respectively, and/or a lightchain variable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:25, SEQ ID NO:26, and SEQ ID NO:27, respectively. In someembodiments, the antibody comprises a light chain variable regioncomprising SEQ ID NO:14 and/or a heavy chain variable region comprisingSEQ ID NO:18. In some embodiments, an antibody comprising the CDRsdescribed in this paragraph is used in combination with another of theantibodies described in this application, thereby inhibiting polymeraseactivity in a DNA polymerase. For example, in some embodiments, anantibody as described above in this paragraph is combined with anantibody comprising a heavy chain variable region comprising CDR1, CDR2,and CDR3 as follows: SEQ ID NO:34, SEQ ID NO:35, and SEQ ID NO:36,respectively, and/or a light chain variable region comprising CDR1,CDR2, and CDR3 as follows: SEQ ID NO:37, SEQ ID NO:38, and SEQ ID NO:39,respectively.

In some embodiments, an antibody is provided comprising a heavy chainvariable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively, and/or a lightchain variable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. In someembodiments, the antibody comprises a light chain variable regioncomprising SEQ ID NO:15 and/or a heavy chain variable region comprisingSEQ ID NO:19. The inventors have found that antibodies comprising theabove-described CDRs are particularly effective in inhibiting 3′-5′exonuclease activities in polymerases comprising such activities. Insome embodiments, for an antibody comprising the CDRs described in thisparagraph is used in combination with another of the antibodiesdescribed in this application, thereby inhibiting polymerase and/or3′-5′ exonuclease activities. For example, in some embodiments, anantibody as described above in this paragraph is combined with anantibody comprising a heavy chain variable region comprising CDR1, CDR2,and CDR3 as follows: SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24,respectively, and/or a light chain variable region comprising CDR1,CDR2, and CDR3 as follows: SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:27,respectively.

In some embodiments, an antibody is provided comprising a heavy chainvariable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:34, SEQ ID NO:35, and SEQ ID NO:36, respectively, and/or a lightchain variable region comprising CDR1, CDR2, and CDR3 as follows: SEQ IDNO:37, SEQ ID NO:38, and SEQ ID NO:39, respectively. In someembodiments, the antibody comprises a heavy chain variable regioncomprising SEQ ID NO:16 and/or a light chain variable region comprisingSEQ ID NO:20. In some embodiments, the antibody comprises a heavy chainvariable region comprising SEQ ID NO:17 and/or a light chain variableregion comprising SEQ ID NO:21.

The present invention also provides for a polynucleotide encoding aprotein (including but not limited to an antibody of antigen fragmentthereof) comprising one, two, or three CDRs of any of the heavy or lightchains as described above. In some embodiments, the encoded proteincomprises a heavy or light variable region, e.g., as set forth in any ofSEQ ID NOs:14, 15, 16, 17, 18, 19, 20, or 21. In some embodiments, theprotein comprises additional antibody components, and in some cases is acomplete antibody heavy or light chain. In some embodiments, theinvention provides for expression cassettes or vectors comprising theabove-described polynucleotide, as well as host cells (including but notlimited to bacterial, fungal, yeast, insect, or mammalian cells)comprising such expression cassettes or vectors.

The antibodies described herein are useful, for example, in “hot start”amplification or other reactions. Accordingly, in some embodiments, theyare provided in a kit, optionally also comprising a DNA or RNApolymerase or other reagents for use in nucleic acid amplification.

Additives for Improving Efficiency of Amplification Reactions

In certain aspects, it may be desirable to include an additionalcompound as an additive to improve efficiency in amplificationreactions, including but not limited to qPCR. The amplification reactioncomprising the additives can include mixtures comprising polymeraseshaving or lacking 3′-5′ exonuclease activity and optionally including aheterologous DNA binding domain. In some embodiments, inclusion of theadditive is sufficient to increase efficiency of the polymerase by atleast 5, 10, 15, 20, 25, 35, 40, or 50% or more compared to a controlmixture lacking the additive.

In some embodiments, a polymerase of the invention exhibits lowefficiency for certain targets when used in a formulation that includescertain binding dyes (such as, in one non-limiting example, an EvaGreenDNA binding dye). Such low efficiency may in some embodiments result ina delay of Ct values associated with low input DNA concentrations.Methods for measuring efficiency of a particular reaction are known inthe art and described in further detail below.

In some embodiments, the additive is an osmolyte included in anamplification reaction of the invention to improve efficiency. Membersof the osmolyte family have been shown to improve the thermal stabilityof proteins (Santoro, Biochemistry, 1992) as well as decrease DNA doublehelix stability (Chadalavada, FEBS Letters, 1997). In some embodiments,osmolytes are small molecules or compounds which are produced by livingorganisms in response to environmental stresses such as extremetemperatures, dehydration, or salinity and which protect their cellularcomponents and help to maintain optimal cytosolic conditions. Osmolytesof use in the present invention may include without limitationsarcosine, trimethylamine N-oxide (TMAO), dimethylsulfoniopropionate,and trimethylglycine. Sarcosine is chemically similar to betaine, achemical which has been shown to improve conventional PCR (Henke,Nucleic Acids Research, 1997).

In conventional uses of osmolytes, the stabilizing effects of suchcompounds are generally observed at relatively high concentrations(>1M). However, in methods of the present invention, millimolarconcentrations of osmolytes have been found to be effective forimproving the reaction efficiency of amplification reactions such asqPCR. Without being bound by a mechanism of action, it is possible thatthe improvement in efficiency is the result of improving theaccessibility of the DNA polymerase to the targeted region of the DNAtemplate for reactions that contain low concentrations of input DNAsample. In some embodiments, concentrations of about 100 to about 1000mM of osmolytes are used in methods and kits of the present invention.In still further embodiments, concentrations of about 50 to about 700,about 100 to about 600, about 150 to about 500, about 200 to about 400mM, and about 300 to about 350 mM osmolytes are used in methods and kitsof the invention. In some embodiments, the osmolyte used in methods,reaction mixtures, and kits of the invention is sarcosine (optionally atthe above-listed concentrations). As shown in FIG. 1, addition ofsarcosine improved the efficiency of the amplification reaction ascompared to control lacking sarcosine.

In some embodiments, particularly in the amplification of low-copytarget nucleic acids, efficiency decreases due to the binding ofpolymerase to non-primed double-stranded nucleic acid targets. Bindingof the polymerase to the double-stranded targets will prevent thosetargets from denaturation, hybridizing to primers, and undergoing anamplification reaction. To improve the specificity of the polymerase forprimed templates, in some embodiments methods of the invention utilizeheparin. Heparin molecules, which are negatively charged, can beincluded in the reaction mixture to mimic the electrostatic property ofdouble stranded nucleic acids. The addition of heparin can, withoutbeing limited to a mechanism of action, prevent excess polymerase frombinding to the double-stranded template until a single-strandedprimed-template becomes available. In some exemplary embodiments,heparin is used in methods and kits of the invention at concentrationsof about 50 to about 750 pg/μl. In further exemplary embodiments,heparin is used in methods and kits of the invention at concentrationsof about 75 to about 700, about 100 to about 600, about 125 to about500, about 150 to about 400, about 175 to about 300, and about 200 toabout 250 pg/μl. Other molecules known in the art can be used in asimilar manner to prevent non-specific binding of the polymerase tonon-primed double-stranded template.

Amplification Reactions Utilizing Compositions of the Invention

As discussed herein, the present invention provides differentcompositions, including hybrid polymerases, hot-start antibodies andreaction additives, for use in nucleic acid amplification reactions.Such amplification reactions include without limitation polymerase chainreaction (PCR), DNA ligase chain reaction (LCR), QBeta RNA replicase,and RNA transcription-based (such as TAS and 3 SR) amplificationreactions as well as others known to those of skill in the art.Polymerase chain reactions that can be conducted using the compositionsdescribed herein include without limitation reverse-transcription PCR(rt-PCR) and quantitative PCR (qPCR).

As will be appreciated, any combination of the different componentsdescribed herein is encompassed by the present invention, as areamplification reactions utilizing any combination of differentcomponents of the invention. For example, amplification reactions of theinvention may utilize hybrid polymerases comprising one or mutationsthat remove or completely abolish exonuclease activity, particularly3′-5′ exonuclease activity. Such amplification reactions may furtherutilize such mutant hybrid polymerases combined with hot-startantibodies. Some amplification reactions of the invention may utilizemutant hybrid polymerases comprising the D141A/E143A double pointmutation combined with additives such as sarcosine or heparin. Someamplification reactions of the invention may also utilize mutant hybridpolymerases lacking 3′-5′ exonuclease activity combined with hot-startantibodies and with additives such as sarcosine or heparin. Furthercombinations are easily identified by one of skill in the art using thedisclosure provided herein.

Amplification reactions, such as polymerase chain reaction (PCR)methods, show an improved efficiency and specificity when compositionsof the invention are components of such reactions. Typically,“efficiency” as discussed herein is indicated by the amount of productgenerated under given reaction conditions. For example, in efficientreal-time PCR reactions, the PCR product should double at every cycle.As is known in the art, the efficiency of different kinds ofamplification reactions can be calculated using different methods. Forexample, the exponential amplification of PCR is generally determinedusing the equationX _(n) =X _(o)*(1+E _(x))^(n),  (I)where X_(n) is the number of target molecules at cycle n, X_(o) is theinitial number of target molecules, and E_(x) is the efficiency of thetarget amplification and n is the number of cycles.

Improvements in efficiency and specificity due to certain aspects of thepresent invention can be identified and quantified using assays known inthe art and described in further detail below.

In some embodiments, dye-based qPCR detection methods are used tomonitor amplification reactions utilizing components of the invention.Such detection methods generally rely on monitoring the increase influorescence signal due to the binding of DNA-binding dye to theamplified DNA. For example, SYBR Green I, a commonly used fluorescentDNA binding dye, binds all double-stranded DNA and detection ismonitored by measuring the increase in fluorescence throughout thecycle. SYBR Green I has an excitation and emission maxima of 494 nm and521 nm, respectively.

In other embodiments, probe-based qPCR detection methods are used tomonitor amplification reactions utilizing components of the invention.Such detection methods generally rely on the sequence-specific detectionof a desired PCR product. Unlike dye-based qPCR methods that detect alldouble-stranded DNA, probe-based qPCR utilizes a fluorescent-labeledtarget-specific probe, which detects specific sequences in the amplifiedDNA.

Ct Determination

In qPCR applications utilizing dual-labeled fluorogenic probes (such asTaqMan® probes from Applied BioSystems), the amount of cleavage productgenerated by 5′-3′ exonuclease activity during the reaction isdetermined based on cycle threshold (Ct) value, which represents thenumber of cycles required to generate a detectable amount of DNA. InqPCR applications utilizing dual-labeled fluorogenic probes (such asTaqMan® probes from Applied BioSystems), the amount of cleavage productgenerated is monitored by fluorescence of a dye which is released fromthe fluorogenic probe through the polymerase's 5′ exonuclease activity.The number of cycles required to generate a detectable amount of DNA, asmonitored by dye fluorescence, is referred to as the cycle threshold(Ct). As the amount of formed amplicon increases, the signal intensityincreases to a measurable level and reaches a plateau in later cycleswhen the reaction enters into a non-logarithmic phase. By plottingsignal intensity versus the cycle number during the logrithmic phase ofthe reaction, the specific cycle at which a measurable signal isobtained can be deduced and used to calculate the quantity of the targetbefore the start of the PCR. Exemplary methods of determining Ct aredescribed in, e.g., Heid et al. Genome Methods 6:986-94, 1996. The Ctvalue represents the number of cycles required to generate a detectableamount of DNA (a “detectable” amount of DNA is typically 2×, 5×, 10×,100× or more above background). An efficient polymerase may be able toproduce a detectable amount of DNA in a smaller number of cycles by moreclosely approaching the theoretical maximum amplification efficiency ofPCR. Accordingly, a lower Ct value for a given amount of input DNAtemplate reflects a greater amplification efficiency for the enzyme.

Assays to Evaluate Processivity and Efficiency

Polymerase processivity can be measured by a variety of methods known tothose of ordinary skill in the art. Polymerase processivity is generallydefined as the number of nucleotides incorporated during a singlebinding event of a modifying enzyme to a primed template. For example, a5′ FAM-labeled primer is annealed to circular or linearized DNA to forma primed template. In measuring processivity, the primed templateusually is present in significant molar excess to the polymerase so thatthe chance of any primed template being extended more than once by thepolymerase is minimized. The primed template is therefore mixed with thepolymerase at a ratio such as approximately 4000:1 (primed DNA:DNApolymerase) in the presence of buffer and dNTPs. MgCl2 is added toinitiate DNA synthesis. Samples are quenched at various times afterinitiation, and analyzed on a sequencing gel. At a polymeraseconcentration where the median product length does not change with timeor polymerase concentration, the length corresponds to the processivityof the enzyme. The processivity of a polymerase of the invention is thencompared to the processivity of a wild type enzyme.

Improvement in processivity can be reflected in the decrease in saltsensitivity of the polymerase. In some embodiments, a polymerase of thepresent invention tolerates higher salt concentration as itsprocessivity is improved due to the presence of the dsDNA bindingdomain. For example, a PCR analysis can be performed to determine theamount of product obtained in a reaction using a polymerase of thepresent invention compared to a wild type polymerase in reactionmixtures with varied salt concentration. While both polymerases mayproduce similar amount of product at 50 mM KCl, the polymerase of thepresent invention is expected to outperform the wild type polymerase athigher KCl concentrations, e.g. 80 mM, 100 mM, etc.

Efficiency can be demonstrated by measuring the ability of an enzyme toproduce product. Increased efficiency can be demonstrated by measuringthe increased ability of an enzyme to produce product. Such an analysismeasures the stability of the double-stranded nucleic acid duplexindirectly by determining the amount of product obtained in a reaction.For example, a PCR assay can be used to measure the amount of PCRproduct obtained with a short, e.g., 12, or 18-20 nucleotide in length,primer annealed at an elevated temperature, e.g., 50° C. In thisanalysis, enhanced efficiency is shown by the ability of a modified orimproved polymerase to produce more product in a PCR reaction using theshort primer annealed at 50° C. than a wild-type polymerase under thesame conditions.

Long PCR may be used as another method of demonstrating enhancedprocessivity and efficiency. For example, an enzyme with enhancedprocessivity and efficiency typically allows the amplification of a longamplicon (>5 kb) in a shorter extension time compared to an enzyme withrelatively lower processivity and efficiency.

Other methods of assessing efficiency of the polymerases of theinvention can be determined by those of ordinary skill in the art usingstandard assays of the enzymatic activity of a given modificationenzyme.

Primer/template specificity is the ability of an enzyme to discriminatebetween matched primer/template duplexes and mismatched primer/templateduplexes. Specificity can be determined, for example, by comparing therelative yield of two reactions, one of which employs a matched primer,and one of which employs a mismatched primer. An enzyme with increaseddiscrimination will have a higher relative yield with the matched primerthan with the mismatched primer, i.e., the ratio of the yield in thereaction using the matched primer vs. the reaction using the mismatchedprimer is about 1 or above. This ratio can then be compared to the yieldobtained in a parallel set of reactions employing a wild typepolymerase. Reactions utilizing methods and compositions of theinvention will in may embodiments exhibit at least a 2-fold, often3-fold or greater increase in the ratio relative to reactions utilizingwild-type polymerases and/or standard reaction conditions.

Reaction Mixtures of the Invention

The present invention also provides reaction mixtures comprising thepolymerases of the invention, the antibodies of the invention, theadditives of the invention, or any combination or two or three thereof.Optionally, the antibody is complexed with the polymerase. The reactionmixtures can optionally comprise one or more dNTPs, one or moreoligonucleotides, a biological sample comprising a target nucleic acid,and/or a double stranded DNA binding dye. Any one, two or more of theantibodies as described herein can be included in the reaction mixture.

Kits of the Invention

In one aspect, the present invention provides kits for conductingnucleic acid amplification reactions. In some embodiments, such kitsinclude polymerases, and optionally dNTPs, and at least one buffer. Suchkits may also include stabilizers and other additives (e.g., heparinand/or sarcosine) to increase the efficiency of the amplificationreactions. Such kits may also include one or more primers as well asinstructions for conducting nucleic acid amplification reactions usingthe components of the kits.

In a further aspect, the present invention provides kits that includecomponents that improve the efficiency and specificity of nucleic acidamplification reactions over reactions conducted using conventionalreaction conditions and reactants. Such additional components aredescribed further herein and include without limitation hybridpolymerases, hot-start antibodies, and/or additives such as sarcosineand heparin.

In one embodiment, kits of the invention include a hybrid polymerasecomprising a mutation in its polymerase domain that reduces or abolishesthe polymerase's 3′-5′ exonuclease activity. In a further embodiments,kits of the invention include a hybrid polymerase with an amino acidsequence substantially identical to SEQ ID NO: 2.

In some embodiments, the polymerase will be fused to a DNA bindingdomain. In some embodiments, the DNA binding domain will be an Ssobinding domain. In some embodiments, the Sso binding domain is identicalor substantially identical to SEQ ID NO:3.

In a still further embodiments, kits of the invention include a hybridpolymerase complexed with one or more specific monoclonal antibodies toachieve “hot-start” capabilities. Such monoclonal antibodies may in someexemplary embodiments comprise light-chain variable regions withnucleotide sequences of about 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 10-13. Suchmonoclonal antibodies may in further exemplary embodiments compriseheavy-chain variable regions with nucleotide sequences of about 75%,80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity toany one of SEQ ID NOs: 6-9. Such monoclonal antibodies may in stillfurther exemplary embodiments comprise heavy-chain variable regions withamino acid sequences of about 75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%,98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 14-17and/or light-chain variable regions with amino acid sequences of about75%, 80%, 85%, 90%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to any one of SEQ ID NOs: 18-21.

In some embodiments, the antibody comprises a heavy chain variableregion comprising CDR1, CDR2, and CDR3 as follows: SEQ ID NO:22, SEQ IDNO:23, and SEQ ID NO:24, respectively, and/or a light chain variableregion comprising CDR1, CDR2, and CDR3 as follows: SEQ ID NO:25, SEQ IDNO:26, and SEQ ID NO:27, respectively. In some embodiments, the antibodycomprises a heavy chain variable region comprising CDR1, CDR2, and CDR3as follows: SEQ ID NO:28, SEQ ID NO:29, and SEQ ID NO:30, respectively,and/or a light chain variable region comprising CDR1, CDR2, and CDR3 asfollows: SEQ ID NO:31, SEQ ID NO:32, and SEQ ID NO:33, respectively. Insome embodiments, the antibody comprises a heavy chain variable regioncomprising CDR1, CDR2, and CDR3 as follows: SEQ ID NO:34, SEQ ID NO:35,and SEQ ID NO:36, respectively, and/or a light chain variable regioncomprising CDR1, CDR2, and CDR3 as follows: SEQ ID NO:37, SEQ ID NO:38,and SEQ ID NO:39, respectively.

In still further embodiments, kits of the invention include optimizedbuffer (Tris-HCl, pH 9.0), KCl, (NH4)2SO4, stabilizer, detergent, dNTPs,MgCl2, and DMSO.

In still further embodiments, kits of the invention include doublestranded DNA binding dyes. Such double stranded DNA binding dyes caninclude without limitation: EvaGreen and SYBR Green, as well as anyother double stranded DNA binding dyes known in the art.

In a still further embodiment, kits of the invention include additivesto increase the specificity and efficiency of nucleic acid amplificationreactions. Such additives include without limitation sarcosine andheparin.

It will be appreciated that kits of the invention also encompass anycombination of the above-described components.

In some aspects, instructions included with kits of the invention willinclude typical amplification protocols that include the followingsteps:

-   -   95-98° C. for about 30 seconds to about 2 minutes    -   40 cycles (95-98° C. for about 1 to about 5 seconds, 60° C. for        about 1 to about 5 seconds, detection step)    -   Melting cycle at about 60° C. to about 95° C.

It will be appreciated that the above exemplary protocol can be variedusing parameters well known in the art to optimize nucleic acidamplification reactions to optimize the conditions for efficiency andspecificity for different target nucleic acids. For example,amplification of longer target nucleic acids may require longerincubation times and/or higher temperatures for efficient and specificamplification.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes.

EXAMPLES Example 1: Comparing Primer-Dimer Formation in ReactionsUtilizing Hybrid Polymerases with and without Exonuclease Activity

The tendency of primer dimer formation was compared for two differentpolymerases. One is a hybrid polymerase enzyme (A), which has both 5′-3′polymerization activity and 3′-5′ exonuclease activity forproof-reading, and the other is a mutant hybrid (B), which contains twopoint mutations (E141A, D143A—SEQ ID NO:2) that abolish the 3′-5′exonuclease activity without affecting the polymerase activity. Foursets of primers (a, b, c, d) were designed to present differentscenarios of primer dimer formation. The characteristics of these foursets of primers were as follows:

-   -   (a) The two primers can form three G-C base pairs near but not        at the 3′ end of each primer.    -   (b) There is no tendency of primer-primer annealing based on the        primer sequences.    -   (c) The two primers can form 3 base pairs at the 3′ end.    -   (d) The two primers can form three A-T base pairs near but not        at the 3′ end of each primer.

Each of these primer pairs were pre-incubated in a reaction buffer withpolymerase (A) or (B) at 45° C. for 30 minutes to promote primer dimerformation and extension, followed by a qPCR amplification protocol, sothat the primer dimers formed during the pre-incubation step werefurther amplified. The numbers indicated in Table I are the Ct(threshold cycle) values from the qPCR amplification, and they correlatewith the amount of primer dimer formed during the pre-incubation step,i.e. the smaller the Ct value the higher the amount of primer dimerformation (higher primer dimer formation is less favorable orefficient). In the experiment producing the data in Table 1, bothenzymes were pre-bound to corresponding hot-start antibodies to fullyinhibit the polymerase activity at low temperature.

Primer pair (a): Polymerase (A) resulted in ˜14 cycle earlier Ctcompared to polymerase (B). A 14 cycle difference correlatesto >10000-fold difference in the amount of primer-dimer formed. It islikely that the difference arises because the 3′-5′ exo nucleaseactivity of polymerase (A) cleaves the 3′ mismatched region, and leavesa fully complimentary 3′ end between the two primers, which can then beused as substrate for the polymerase activity. Conversely, polymerase(B) which lacks the 3′-5′ exonuclease activity, is not able to cleavethe 3′ mismatched region, and the 3′-mismatched structure cannot then beefficiently utilized by the polymerase activity for extension, thusresulting in much lower level of primer-dimer formation. In thissituation, polymerase (B) is the preferred enzyme.

Primer pair (b): when the primers have no tendency of annealing witheach other, there is a low tendency of primer dimer formation with bothpolymerases.

Primer pair (c): When the primers have the tendency to formcomplementary sequences at the 3′ end, polymerase (A) gave significantlylater Ct than polymerase (B). The delay in Ct associated with polymerase(A) is likely due to the digestion of the 3′ terminus of the primers byits 3′-5′ exo nuclease activity, which eliminates the sequences thathave the potential of forming base pairs with another primer molecule.Although, in general, it is desirable to have delayed Ct value withrespect to amplification of primer dimers, in this particular case, theproperty of the primers are altered, both in length and in 3′ sequence,which could lead to undesirable amplifications in the presence oftemplate.

Primer pair (d): this is similar to primer pair (a) except the 3′ basepairs are not as stable as those in primer pair (a). Identical Ct valuesare observed with both polymerases.

TABLE 1 (a) IL1B primers (b) BA primers (c) GAPD primers (d) 18s primers

Enzyme (A) 21.6 ± 0.3 36.5 ± 1.5 44.5 ± 0.9 33.1 ± 0.4 mutant Enzyme(B)35.3 ± 1.3 34.6 ± 1.0 37.2 ± 3.2 33.4 ± 1.0 dCt (A-B) −13.7 1.9 7.3 −0.3

To evaluate whether the difference observed between the two polymeraseswas due to the presence of the antibodies or the differences in theantibodies used, a similar assay was conducted using a hybrid and amutant (exonuclease deficient) hybrid as described above that are notcomplexed with the antibodies. Four sets of primers were used (see Table2), with set A and B containing fully complementary base pairs at the 3′end, and C containing base pairs near the 3′ end followed by one basemismatch at the 3′ end. With set A and B, the hybrid polymerase gave 5-8cycle delay in Ct compared to the mutant hybrid. With set C of primers,significant earlier Ct value is obtained with hybrid polymerase comparedto mutant hybrid polymerase. The overall results obtained using hybridpolymerase and mutant hybrid polymerase alone are similar to thatobtained using hot start antibodies with the polymerases. Therefore, itis likely that the difference observed between the two polymerases isdue to the difference in the exonuclease activity rather than thepresence of antibodies.

In summary, a polymerase lacking exonuclease activity such as the mutanthybrid described above is an effective enzyme for qPCR applications toavoid primer-dimer formation/amplification in situations where primershave the tendency of forming base pairs near the 3′ end and leavingmismatched bases at the 3′ end, as well as to eliminate alteration ofprimer length/sequence in situations where primers form base pairs atthe 3′ end.

TABLE 2 A B C

hybrid 28.72 15.5 16.2 mutant hybrid 20.90 10.17 28.85dCt(hybrid-mutant) 7.82 5.33 −12.65

Example 2: Comparing Amplification Efficiency of Hybrid Polymerases withand without Exonuclease Activity

Additional experiments were conducted to directly compare hybridpolymerases complexed with hot-start antibodies to mutant hybridpolymerases lacking exonuclease activity, which were also complexed withthe same hot-start antibodies. The same formulation composition was usedwith equal concentrations of either the hybrid or the mutant hybrid. Theperformance of the two enzymes was compared in the following twoaspects: (1) ability to work with wide range of annealing/extensiontemperatures, and (2) ability to quantify wide range of input DNAtemplate. Annealing and extension temperature range used is 56.6° C. to66.6° C. The amplicon is from human beta-actin gene, and the input DNAtemplate is 25, 250, and 2500 pg human genomic DNA per reaction. Asshown in FIG. 3, the amplification curves and the Ct values obtainedwith the mutant hybrid (right panel) showed less deviation as theannealing/extension temperature changes from 56.6° C. to 66.6° C. Incontrast, significant spread in the amplification curve and thus Ctvalue is observed with the hybrid polymerase (left panel) with respectto annealing/extension temperature change. In addition, hot-start hybridgave much lower and more scattered amplification efficiency over therange of annealing/extension temperature tested, whereas hot-startmutant hybrid gave consistently higher efficiency and is notsignificantly affected by change of the annealing/extension temperature.(see Table 3).

TABLE 3 Annealing/extension Efficiency of hybrid Efficiency of mutanthybrid temp polymerase polymerase 66.6 C. 43.3% 94.8% 66.1 C. 46.6%90.2% 64.9 C. 52.7% 98.8% 63.0 C. 56.1% 92.1% 60.6 C. 72.7% 92.3% 58.6C. 62.3% 88.6% 57.3 C. 59.4% 93.0% 56.6 C. 67.2% 95.0%

Example 3: Primer-Dimer Assays for Assessment of Hot-Start Antibodies

As described earlier, hot start DNA polymerases are used in PCR andespecially real-time PCR to minimize or eliminate non-specificamplifications, as the latter is often performed in the presence of lowquantities of input DNA template and is more prone to nonspecificamplifications. A primer dimer assay was developed to allow quantitativeassessment of whether the hybrid polymerase or mutant polymerasedescribed above, complexed with the appropriate antibodies, are suitablefor real-time PCR applications. In this assay, a pair of primers wasdesigned to form 3 to 5 complementary base pairs at the 3′ end with eachother. The assay consists of two steps: (1) the primer pair wasincubated in a qPCR mix with all components required for DNA replicationat 37-45° C. for 30 minutes; (2) the reaction in the first step willcontinue using standard qPCR cycling and detection protocol and allowedto be amplified. If the DNA polymerase was active at 37-45° C., then theprimers would be extended off each other during the first step togenerate templates for amplification during the second step. If the DNApolymerase was not active or has a significantly lower activity in thistemperature range, then only low level of primer extension would occurduring the first step, and the amplification during the second step willresult in delayed Ct. Therefore, by comparing the Ct values obtainedwith the DNA polymerase alone versus that with the DNA polymerasecomplexed with the antibodies, it was possible to assess the benefit ofhot start antibodies in preventing/minimizing primer dimer formation.Table 4 summarizes the results obtained using three primer sets thatdiffer in tendency of forming base pairs at or near the 3′ end. Overall,the addition of hot start antibodies resulted in 5-11 cycles of Ct delayin primer dimer amplification compared to the reactions without theaddition of the antibodies. The extent of the benefit of using hot startantibodies was similar between the two polymerases.

TABLE 4 c a 4 bp match near 3′ 3 bp b end match at 5 bp match w/ 1mismatch at 3′ 3′end at 3′end end hybrid polymerase 28.72 15.5 16.2hybrid polymerase 35.05 26.43 22.46 complexed with antibodies dCt(hybrid− (hybrid + −6.33 −10.93 −6.26 antibodies)) mutant hybrid 20.90 10.1728.85 polymerase mutant hybrid polymerase complexed 26.03 16.17 37.82with antibodies dCt(mutant hybrid − −5.13 −6.00 −8.97 (mutant hybrid +antibodies))

Example 4: Determining Effective Concentration of Sarcosine

To determine the effective concentration range of sarcosine, differentconcentrations of sarcosine (from 20 mM to 540 mM) was added to a qPCRmix comprising a mutant hybrid polymerase lacking 3′-5′ exonucleaseactivity, as described above. The effect of sarcosine on qPCR reactionefficiency was evident with sarcosine concentrations as low as 20 mM.Concentration greater than 40 mM gave optimal and similar performance(Table 6). This is in contrast to published results which showed optimalosmolyte effects at concentrations above 1M, and suggests that themechanism of action of sarcosine in the present invention differs frompublished observations of the effects of osmolytes. Similar results wereobserved with a Sso7-Taq fusion polymerase.

TABLE 6 CBP amplicon (50 ng-50 pg gDNA) Slope/R2 Amplificationefficiency  0 mM −4.466/0.980 67.5%  20 mM −3.688/0.997 86.7%  40 mM−3.352/0.997 98.78%   60 mM −3.370/0.991 98.0%  80 mM −3.386/0.987 97.4%100 mM −3.329/0.998  104% 100 mM −3.228/0.998  104% 200 mM −3.341/0.99199.2% 300 mM −3.177/0.995  106% 400 mM −3.227/0.998  104% 540 mM−3.150/0.995  108%

Example 5: Effect of Heparin

Heparin is a negatively charged polymer that mimics the electrostaticproperty of dsDNA, and is commonly used for either purifying DNA bindingproteins or as a nonspecific competitor for DNA binding proteins. Theaddition of heparin may prevent the excess DNA polymerase from bindingto the double-stranded template until a single-stranded primed-templatebecomes available.

A CBP amplicon was amplified in qPCR reactions from two different inputamounts (5 ng and 0.5 ng) of human genomic DNA template with qPCRformulation comprising a hybrid polymerase, in which 0, 10, 20, 100, 200μg/μl of heparin was added (FIG. 2, Table 7). If the amplificationefficiency is 100%, the expected Ct difference corresponding to 10-folddifference in input template concentration is 3.33. When no heparin isadded, the ΔCt between the 5 ng and 0.5 ng reactions was 4.5, whichcorresponds to 67% amplification efficiency. When 100 ng of heparin wasadded, the ΔCt between the 5 ng and 0.5 ng reactions was 3.4corresponding to 97% amplification efficiency, which was a significantimprovement in comparison to no-heparin reactions.

TABLE 7 no 10 pg/ul 20 pg/ul 100 pg/ul 200 pg/ul Heparin Heparin HeparinHeparin Heparin 5 ng gDNA 25.8 25.8 25.8 25.3 25.2 0.5 ng gDNA 30.3 30.329.4 28.7 28.8 ΔCt (0.5 ng-5 ng) 4.50 4.50 3.60 3.40 3.60 Efficiency 67%67% 90% 97% 90%

SEQUENCE LISTING (A) SEQ ID NO: 1 ATGATCCTGGATGCTGACTACATCACTGAAGAAGGCAAACCGGTTATCCGTCTGTTCAAAAAAGAGAACGGCGAATTTAAGATTGAGCATGATCGCACCTTTCGTCCATACATTTACGCTCTGCTGAAAGATGATTCTAAGATTGAGGAAGTTAAAAAAATCACTGCTGAGCGCCATGGCAAGATTGTTCGTATCGTTGATGCGGAAAAGGTAGAAAAGAAATTTCTGGGCAGACCAATCACCGTGTGGAGACTGTATTTCGAACATCCACAAGATGTTCCGACTATTCGCGAGAAAATTCGCGAACATTCTGCAGTTGTTGACATCTTCGAATACGATATTCCATTTGCAAAGCGTTACCTCATCGACAAAGGCCTGATACCAATGGAGGGCGATGAAGAACTCAAGCTCCTGGCGTTCGCTATAGCAACCCTCTATCACGAAGGCGAAGAGTTTGGTAAAGGCCCAATTATAATGATCAGCTATGCAGATGAAGAAGAAGCAAAGGTGATTACTTGGAAAAAAATAGATCTCCCATACGTTGAGGTTGTATCTTCCGAGCGCGAGATGATTAAGCGCTTTCTCAAAATTATCCGCGAGAAGGATCCGGACATTATCATTACTTATAACGGCGACTCTTTTGACCTCCCATATCTGGCGAAACGCGCAGAAAAACTCGGTATTAAACTGACTATCGGCCGTGATGGTTCCGAGCCGAAGATGCAGCGTATCGGCGATATGACCGCTGTAGAAGTTAAGGGTCGTATCCATTTCGACCTGTATCATGTAATTCGTCGTACTATTAACCTCCCGACTTACACTCTCGAGGCTGTATATGAAGCAATTTTTGGTAAGCCGAAGGAGAAGGTATACGCCGATGAGATTGCAAAGGCGTGGGAAACCGGTGAGGGCCTCGAGCGTGTTGCAAAATACTCCATGGAAGATGCAAAGGCGACTTATGAACTCGGCAAAGAATTCTTCCCAATGGAAGCTCAGCTCTCTCGCCTGGTTGGCCAACCACTGTGGGATGTTTCTCGTTCTTCCACCGGTAACCTCGTAGAGTGGTTTCTCCTGCGCAAAGCGTACGAACGCAACGAACTGGCTCCGAACAAGCCAGATGAACGTGAGTATGAACGCCGTCTCCGCGAGTCTTACGCTGGTGGCTTTGTTAAAGAGCCAGAAAAGGGCCTCTGGGAAAACATCGTGTCCCTCGATTTTCGCGCTCTGTATCCGTCTATTATCATTACCCACAACGTGTCTCCGGATACTCTCAACCGCGAGGGCTGCAGAAACTATGATGTTGCTCCGGAAGTAGGCCACAAGTTCTGCAAGGACTTCCCGGGCTTTATTCCGTCTCTCCTGAAACGTCTGCTCGATGAACGCCAAAAGATTAAGACTAAAATGAAGGCGTCCCAGGATCCGATTGAAAAAATAATGCTCGACTATCGCCAAAGAGCGATTAAAATCCTCGCAAACTCTTATTACGGCTATTATGGCTATGCAAAAGCACGCTGGTACTGTAAGGAGTGTGCTGAGTCCGTTACTGCTTGGGGTCGCGAATACATCGAGTTCGTGTGGAAGGAGCTCGAAGAAAAGTTTGGCTTTAAAGTTCTCTACATTGACACTGATGGTCTCTATGCGACTATTCCGGGTGGTAAGTCTGAGGAAATTAAGAAAAAGGCTCTAGAATTTGTGGATTACATTAACGCGAAGCTCCCGGGTCTCCTGGAGCTCGAATATGAAGGCTTTTATAAACGCGGCTTCTTCGTTACCAAGAAGAAATATGCGCTGATTGATGAAGAAGGCAAAATTATTACTCGTGGTCTCGAGATTGTGCGCCGTGATTGGAGCGAAATTGCGAAAGAAACTCAAGCTAGAGTTCTCGAGGCTATTCTCAAACACGGCAACGTTGAAGAAGCTGTGAGAATTGTAAAAGAAGTAACCCAAAAGCTCTCTAAATATGAAATTCCGCCAGAGAAGCTCGCGATTTATGAGCAGATTACTCGCCCGCTGCATGAGTATAAGGCGATTGGTCCGCACGTGGCTGTTGCAAAGAGACTGGCTGCTAAAGGCGTGAAAATTAAACCGGGTATGGTAATTGGCTACATTGTACTCCGCGGCGATGGTCCGATTAGCAACCGTGCAATTCTAGCTGAGGAATACGATCCGAGAAAGCACAAGTATGACGCAGAATATTACATTGAGAACCAGGTGCTCCCGGCGGTACTCCGTATTCTGGAGGGTTTTGGCTACCGTAAGGAAGACCTCCGCTGGCAAAAGACTAAACAGACTGGCCTCACTTCTTGGCTCAACATTAAAAAATCCGGTACCGGCGGTGGCGGTGCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAG TGA (B) SEQ ID NO: 2MILDADYITEEGKPVIRLFKKENGEFKIEHDRTFRPYIYALLKDDSKIEEVKKITAERHGKIVRIVDAEKVEKKFLGRPITVWRLYFEHPQDVPTIREKIREHSAVVDIFEYDIPFAKRYLIDKGLIPMEGDEELKLLAF A I A TLYHEGEEFGKGPIIMISYADEEEAKVITWKKIDLPYVEVVSSEREMIKRFLKIIREKDPDIIITYNGDSFDLPYLAKRAEKLGIKLTIGRDGSEPKMQRIGDMTAVEVKGRIHFDLYHVIRRTINLPTYTLEAVYEAIFGKPKEKVYADEIAKAWETGEGLERVAKYSMEDAKATYELGKEFFPMEAQLSRLVGQPLWDVSRSSTGNLVEWFLLRKAYERNELAPNKPDEREYERRLRESYAGGFVKEPEKGLWENIVSLDFRALYPSIIITHNVSPDTLNREGCRNYDVAPEVGHKFCKDFPGFIPSLLKRLLDERQKIKTKMKASQDPIEKIMLDYRQRAIKILANSYYGYYGYAKARWYCKECAESVTAWGREYIEFVWKELEEKFGFKVLYIDTDGLYATIPGGKSEEIKKKALEFVDYINAKLPGLLELEYEGFYKRGFFVTKKKYALIDEEGKIITRGLEIVRRDWSEIAKETQARVLEAILKHGNVEEAVRIVKEVTQKLSKYEIPPEKLAIYEQITRPLHEYKAIGPHVAVAKRLAAKGVKIKPGMVIGYIVLRGDGPISNRAILAEEYDPRKHKYDAEYYIENQVLPAVLRILEGFGYRKEDLRWQKTKQTGLTSWLNIKKSGTGGGGATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQ MLEKQKK*(C) SEQ ID NO: 3 ATVKFKYKGEEKEVDISKIKKVWRVGKMISFTYDEGGGKTGRGAVSEKDAPKELLQMLEKQKK (D) SEQ ID NO: 4GCAACCGTAAAGTTCAAGTACAAAGGCGAAGAAAAAGAGGTAGACATCTCCAAGATCAAGAAAGTATGGCGTGTGGGCAAGATGATCTCCTTCACCTACGACGAGGGCGGTGGCAAGACCGGCCGTGGTGCGGTAAGCGAAAAGGACGCGCCGAAGGAGCTGCTGCAGATGCTGGAGAAGCAGAAAAAG (E) SEQ ID NO: 5 GTGGGG(A) Heavy-chain variable region - nucleotide sequence >M13-vh (SEQ ID NO: 6)GAAGTACAGCTGGAGCAGTCAGGGGCTGAGCTGGTGAGGCCTGGGTCCTCAGTGAAGATTTCCTGCAAGGCTTCTGGCTATGTTTTCAGTACCTACTGGATGAACTGGGTGAAGCAGAGGCCTGGACAGGGTCTTGAGTGGATTGGACAGATTTATCCTGGAGATGGTGGTGTTAACTACAATGGAGAGTTCAAGGCTAGGGCCACACTGACTGCAGACAGATCCTCCAGCACAGCCTACATGCAGCTCAGGAGCCTAACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGTTCTTCTTACTACGGTGGTAGTTCCGTCTCCTGGCTTGCTTACTGGGGCCAAGGGACTCTGGTCTCTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATA >M15- vh (SEQ ID NO: 7)GAAGTAAAGCTGGAGGAGTCAGGGGCTGAGTTGGCAAGACCTGGGGCTTCAGTGAAGTTGTCCTGCAAGGCTTCTGGCTACACCTTTACTAGCTACTGGATGCAGTGGGTAAAACAGAGGCCTGGACAGGGTCTGGAATGGATTGGGGCTATTTATCCTGGAGATGGTGAGACTTGGCACACTCAGAAGTTCAAGGGCAAGGCCACATTGACTGCAGATAAATCCTCCAGCACAGCCTACATGCAACTCAGCAGCTTGGAATCTGAGGACTCTGCGGTCTATTACTGTGCAAGAGAAAGGGATAGGTTCGACGGGTCCTGGTTTGCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATA >M22-vh (SEQ ID NO: 8)GAGGTAAAGCTGCAGGAGTCTGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACGTTCACCACCTACTGGATGAACTGGGTTAAGCAGAGGCCTGAGCAAGGCCTTGAGTGGATTGGAAGGATTGATCCTTACGATAGTGACACTCACTACAATCAAAAGTTCAGTGACAAGGCCATATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAAGGGGGGACTATGTCCCCATCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTCTATA >M28-vh (SEQ ID NO: 9)GAGGTGAAGCTGGAGGAGTCAGGGGCTGAGCTGGTGAGGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACGTTCACCACCTACTGGATGAACTGGGTTAAGCAGAGGCCTGAGCAAGGCCTTGAGTGGATTGGAAGGATTGATCCTTACGATAGTGACACTCACTACAATCAAAAGTTCAGTGACAAGGCCATATTGACTGTAGACAAATCCTCCAGCACAGCCTACATGCAACTCAACAGCCTGACATCTGAGGACTCTGCGGTCTATTACTGTTCAAGGGGGGACTATGTCCCCATCTATGCTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACGACACCCCCATCTGTC TATA(B) Light-chain variable region - nucleotide sequence >M13-vl (SEQ ID NO: 10)GACATTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAAGTCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGACTTTATTACTGCTTTCAAGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCA >M15-vl (SEQ ID NO: 11)GACATTGTGATGACACAGACTCCAGCTTCTTTGGCTGTGTCTCTAGGGCAGAGGGCCACCATCTCCTGCAAGGCCAGCCAAAGTGTTGATTATGATGGTGATAGTTATATGAACTGGTACCAACAGAAACCAGGACAGCCACCCAAACTCCTCATCTATGCTGCATCCAATCTAGAATCTGGGATCCCAGCCAGGTTTAGTGGCAGTGGGTCTGGGACAGACTTCATCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATCACTGTCAGCAAAGTAATGAGGATCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCC >M22-vl (SEQ ID NO: 12)GATATTGTGATGACCCAGACTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAATGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATATTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGACGGGCTGATGCTGCACCAACTGTATCC >M28-vl (SEQ ID NO: 13)GATATTGTGATCACACAGTCTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCCTTGTACACAGTAATGGAAACACCTATTTACATTGGTACCTGCAGAGGCCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCAATGGCAGTGGATCAGGGACAGATTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAATTTATTTCTGCTCTCAAAGTACACATATTCCTCGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAGACGGGCTGATGCTGCACCAACTGTATCCA(C) Heavy-chain variable region - putative amino acid sequence >M13-vh (SEQ ID NO: 14)EVQLEQSGAELVRPGSSVKISCKASGYVFSTYWMNWVKQRPGQGLEWIGQIYPGDGGVNYNGEFKARATLTADRSSSTAYMQLRSLTSEDSAVYFCASSSYYGGSSVSWLAYWGQGTLVSVSAAKTTPPSVY >M15-vh (SEQ ID NO: 15)EVKLEESGAELARPGASVKLSCKASGYTFTSYWMQWVKQRPGQGLEWIGAIYPGDGETWHTQKFKGKATLTADKSSSTAYMQLSSLESEDSAVYYCARERDRFDGSWFAYWGQGTLVTVSAAKTTPPSVY >M22-vh (SEQ ID NO: 16)EVKLQESGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSDTHYNQKFSDKAILTVDKSSSTAYMQLNSLTSEDSAVYYCSRGDYVPIYAMDYWGQGTSVTVSSAKTTPPSVY >M28-vh (SEQ ID NO: 17)EVKLEESGAELVRPGASVKLSCKASGYTFTTYWMNWVKQRPEQGLEWIGRIDPYDSDTHYNQKFSDKAILTVDKSSSTAYMQLNSLTSEDSAVYYCSRGDYVPIYAMDYWGQGTSVTVSSAKTTPPSV Y(D) Light-chain variable region - putative amino acid sequence >M13-vl (SEQ ID NO: 18)DIVMTQTPLSLPVSLGDQASISCKSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGLYYCFQGSHVPWTFGGGTKLEIKRADAAPTVS >M15-vl (SEQ ID NO: 19)DIVMTQTPASLAVSLGQRATISCKASQSVDYDGDSYMNWYQQKPGQPPKLLIYAASNLESGIPARFSGSGSGTDFILNIHPVEEEDAATYHCQQSNEDPWTFGGGTKLEIKRADAAPTVS >M22-vl (SEQ ID NO: 20)DIVMTQTPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFNGSGSGTDFTLKISRVEAEDLGIYFCSQSTHIPRTFGGGTKLEIRRADAAPTVS >M28-vl (SEQ ID NO: 21)DIVITQSPLSLPVSLGDQASISCRSSQSLVHSNGNTYLHWYLQRPGQSPKLLIYKVSNRFSGVPDRFNGSGSGTDFTLKISRVEAEDLGIYFCSQSTHIPRTFGGGTKLEIRRADAAPTVSSEQ ID NO: 22 Vh CDR1 M13 T  Y  W  M  N SEQ ID NO: 23 Vh CDR2 M13Q  I  Y  P  G  D  G  G  V  N  Y  N  G  E  F  K  ASEQ ID NO: 24 Vh CDR3 M13 S  S  Y  Y  G  G  S  S  V  S  W  L  A  YSEQ ID NO: 25 Vl CDR1 M13 K  S  S  Q  S  I  V  H  S  N  G  N  T  Y  L  ESEQ ID NO: 26 Vl CDR2 M13 K  V  S  N  R  F SEQ ID NO: 27 Vl CDR3 M13F  Q  G  S  H  V  P  W  T SEQ ID NO: 28 Vh CDR1 M15 S  Y  W  M  QSEQ ID NO: 29 Vh CDR2 M15A  I  Y  P  G  D  G  E  T  W  H  T  Q  K  F  K  GSEQ ID NO: 30 Vh CDR3 M15 E  R  D  R  F  D  G  S  W  F  A  YSEQ ID NO: 31 Vl CDR1 M15 K  A  S  Q  S  V  D  Y  D  G  D  S  Y  M  NSEQ ID NO: 32 Vl CDR2 M15 A  A  S  N  L  E  S SEQ ID NO: 33 Vl CDR3 M15Q  Q  S  N  E  D  P  W  T SEQ ID NO: 34 Vh CDR1 M22 T  Y  W  M  NSEQ ID NO: 35 Vh CDR2 M22R  I  D  P  Y  D  S  D  T  H  Y  N  Q  K  F  S  DSEQ ID NO: 36 Vh CDR3 M22 G  D  Y  V  P  I  Y  A  M  D  YSEQ ID NO: 37 Vl CDR1 M22 R  S  S  Q  S  L  V  H  S  N  G  N  T  Y  L  HSEQ ID NO: 38 Vl CDR2 M22 K  V  S  N  R  F  S SEQ ID NO: 39 Vl CDR3 M22S  Q  S  T  H  I  P  R SEQ ID NO: 40 Vh CDR1 M28 T  Y  W  M  NSEQ ID NO: 41 Vh CDR2 M28R  I  D  P  Y  D  S  D  T  H  Y  N  Q  K  F  S  DSEQ ID NO: 42 Vh CDR3 M28 G  D  Y  V  P  I  Y  A  M  D  YSEQ ID NO: 43 Vl CDR1 M28 R  S  S  Q  S  L  V  H  S  N  G  N  T  Y  L  HSEQ ID NO: 44 Vl CDR2 M28 K  V  S  N  R  F  S SEQ ID NO: 45 Vl CDR3 M28S  Q  S  T  H  I  P  R

What is claimed is:
 1. A reaction mixture comprising: at least oneprimer, an exonuclease deficient hybrid polymerase having a polymerasedomain, wherein said polymerase domain comprises a portion of apolymerase domain from each of two family B parental polymerases, theexonuclease deficient hybrid polymerase further comprising aheterologous DNA binding domain, and a double-stranded DNA binding dye.2. The reaction mixture of claim 1, wherein the reaction mixturecomprises at least two primers that can form a primer dimer.
 3. Thereaction mixture of claim 1, wherein antibodies are bound to the hybridpolymerase.
 4. The reaction mixture of claim 1, wherein the reactionmixture further comprises an additive selected from the group consistingof sarcosine and heparin, in a sufficient amount to improve efficiencyof the amplification reaction by at least 10% compared to a reactionmixture lacking the additive.
 5. The reaction mixture of claim 1,wherein the hybrid polymerase comprises SEQ ID NO:2.
 6. The reactionmixture of claim 1, wherein the heterologous DNA binding domain is atleast 75% identical to SEQ ID NO:3.
 7. The reaction mixture of claim 1,wherein the hybrid polymerase comprises a polypeptide sequence at least90% identical to SEQ ID NO:2.